Compositions and administration of compositions for the treatment of blood disorders

ABSTRACT

The invention relates to novel compositions and to methods for the pulsed administration of compositions to a patient or to cells in vitro for the treatment of human blood disorders. Compositions contain chemical compounds that stimulate the expression of fetal hemoglobin and/or stimulate the proliferation of red blood cells, white blood cells and platelets in patients and ex vivo for reconstitution of hematopoiesis in vivo. These methods are useful to treat or prevent the symptoms associated with anemia, sickle cell disease, thalassemia, blood loss, and other blood disorders. The invention also relates to methods for the pulsed administration of compositions to patients for the treatment and prevention of cell proliferative disorders including deficiencies such as cytopenia and malignancies and for expansion of cells for hematopoietic transplantation. Pulsed administration has been shown to be more effective than continuous therapy in patients tested.

RIGHTS IN THE INVENTION

[0001] This invention was made with support from the United Statesgovernment under grant numbers HL37118 and HL-15157, awarded by theNational Heart, Lung and Blood Institute of the National Institutes ofHealth, and grant number 000831, awarded by the United States Food andDrug Administration, and the United States government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention relates to composition methods for the treatmentand prevention of blood disorders such as anemia, neutropenia,thrombocytopenia, thalassemia and sickle cell disease using suchcompositions. The compositions include C₁-C₄ substituted and/or phenylsubstituted carboylic acids such as dimethyl substitutions ontocarboxylic acids. The methods comprise the administration ofcompositions that stimulate the expression of a globin protein and, inparticular, fetal hemoglobin, or the proliferation or development ofhemoglobin expressing, myeloid cells or megakaryocytic cells.

[0004] 2. Description of the Background

[0005] The major function of red blood cells is to transport oxygen totissues of the body. Minor functions include the transportation ofnutrients, intercellular messages and cytoldnes, and the absorption ofcellular metabolites. Anemia, or a loss of red blood cells or red bloodcell capacity, can be grossly defined as a reduction in the ability ofblood to transport oxygen. Anemia can be measured by determining apatient's red blood cell mass or hematocrit. Hematocrit values areindirect, but fairly accurate measures of the total hemoglobinconcentration of a blood sample. Anemia, as measured by a reducedhematocrit, may be chronic or acute. Chronic anemia may be caused byextrinsic red blood cell abnormalities, intrinsic abnormalities orimpaired production of red blood cells. Extrinsic or extra-corpuscularabnormalities include antibody-mediated disorders such as transfusionreactions and erythroblastosis, mechanical trauma to red cells such asmicro-angiopathic hemolytic anemias, thrombotic thrombocytopenic purpuraand disseminated intravascular coagulation. In addition, infections byparasites such as Plasmodium, chemical injuries from, for example, leadpoisoning, and sequestration in the mononuclear system such as byhypersplenism can result in red blood cell disorders and deficiencies.

[0006] Impaired red blood cell production can occur by disturbing theproliferation and differentiation of the stem cells or committed cells.Some of the more common diseases of red cell production include aplasticanemia, hypoplastic anemia, pure red cell aplasia and anemia associatedwith renal failure or endocrine disorders. Disturbances of theproliferation and differentiation of erythroblasts include defects inDNA synthesis such as impaired utilization of vitamin B₁₂ or folic acidand the megaloblastic anemias, defects in heme or globin synthesis, andanemias of unknown origins such as sideroblastic anemia, anemiaassociated with chronic infections such as malaria, trypanosomiasis,HIV, hepatitis virus or other viruses, and myelophthisic anemias causedby marrow deficiencies.

[0007] Intrinsic abnormalities include both hereditary and acquireddisorders. Acquired disorders are those which have been induced through,for example, a membrane defect such as paroxysmal nocturnalhemoglobinuria. Hereditary disorders include disorders of membranecytoskeleton such as spherocytosis and elliptocytosis, disorders oflipid synthesis such as an abnormally increased lecithin content of thecellular membrane, red cell enzyme deficiencies such as deficiencies ofpyruvate kinase, hexokinase, glutathione synthetase andglucose-6-phosphate dehydrogenase. Although red blood cell disorders maybe caused by certain drugs and immune system disorders, the majority arecaused by genetic defects in the expression of hemoglobin. Disorders ofhemoglobin synthesis include deficiencies of globin synthesis such asthalassemia syndromes and structural abnormalities of globin such assickle cell syndromes and syndromes associated with unstablehemoglobins.

[0008] Mammalian globin gene expression is highly regulated duringdevelopment. The basic structure of the α and β globin genes are similaras are the basic steps in synthesis of α and β globin. There are atleast five human α globin genes located on chromosome 16 including twoadult α globin genes of 141 amino acids that encode identicalpolypeptides which differ only in their 3′-untranslated regions, oneembryonic α gene, zeta (ζ), and at least two pseudo-alpha genes, psizeta (ψβ) and omega alpha (ωα). The human β globin gene cluster includesone embryonic gene, epsilon (ε), two adult beta globin genes, beta (β)and delta (δ), two fetal beta globin genes G-gamma (G-γ) and A-gamma(A-γ), which differ by only one amino acid, and at least one pseudo-betagene, psi beta (ψβ). All are expressed from a single 43 kilobase segmentof human chromosome 11 (E. F. Fritsch et al., Nature 279:598-603, 1979).

[0009] Hemoglobin A comprises four protein chains, two alpha chains andtwo beta chains (α₂β₂), interwoven together, each with its own moleculeof iron and with a combined molecular weight of about 68 kD. Thehemoglobin macromolecule is normally glycosylated and upon absorbingoxygen from the lungs transforms into oxyhemoglobin (HbO₂). There are atleast six distinct forms of hemoglobin, each expressed at various timesduring development. Hemoglobin in the embryo is found in at least threeforms, Hb-Gower 1 (ζ₂β₂), Hb-Gower 2 (α₂γ₂), and Hb-Portand (ζ₂γ₂).Hemoglobin in the fetus comprises nearly totally HbF (α₂γ₂), whereashemoglobin in the adult contains about 96% HbA (α₂β₂), about 3% HbA₂ (α₂δ₂) and about 1% fetal HbF (α₂ γ₂). The embryonic switch of globinexpression from ζ to α and from ε to γ begins in the yolk sac. However,chains of embryonic ζ and ε have been found in the fetal liver andcomplete transition to the fetal form does not occur until late in fetaldevelopment. The fetal switch from γ to β begins later in erythropoeisiswith the amount of γ globin produced increasing throughout gestation. Atbirth, β globin accounts for about 40% of non-α globin chain synthesisand thereafter continues to rapidly increase. Neither the switch fromembryonic to fetal or fetal to adult appears to be controlled throughcell surface or known cytokine interactions. Control seems to reside ina developmental clock with the switch occurring at times determined onlyby the stage of fetal development.

[0010] Defects or mutations in globin chain expression are common. Someof these genetic mutations pose no adverse or only minor consequences tothe person, however, most mutations prevent the formation of an intactor normal hemoglobin molecule through a functional or structuralinability to effectively bind iron, an inability of the chains or chainpairs to effectively or properly interact, an inability of the moleculeto absorb or release oxygen, a failure to express sufficient quantitiesof one or more globin chains or a combination of these malfunctions. Forexample, substitutions of valine for glutamic acid at the sixth positionof the β chain produces HbS and was found to occur in about 30% of blackAmericans. In the HbS heterozygote, only about 40% of total hemoglobinis HbS with the remainder being the more normal HbA.

[0011] Upon deoxygenation, HbS molecules undergo aggregation andpolymerization ultimately leading to a morphological distortion of thered cells which acquire a sickle or holly-leaf shape. Sickling has twomajor consequences, a chronic hemolytic anemia and an occlusion of smallblood vessels that results in ischemic damage to tissues. Further, whenexposed to low oxygen tensions, polymerization converts HbS hemoglobinfrom a free-flowing liquid to a viscous gel. Consequently, the degree ofpathology associated with sickle cell anemia can be correlated with therelative amount of HbS in the patient's system.

[0012] Individuals with severe sickle cell anemia develop no symptomsuntil about five to six months after birth. In these infants it wasdetermined that fetal hemoglobin did not interact with HbS and, as longas sufficient quantities were present, could modulate the effects of HbSdisease. This modulating effect of β globin is also observed with otherβ globin disorders, such as HbC and HbD, and other mutations of the βchain. HbS polymerization is also significantly affected by thehemoglobin concentration of the cell. The higher the HbS concentration,the greater the chances for contact between two or more HbS molecules.Dehydration increases hemoglobin concentration and greatly facilitatessickling.

[0013] To some extent, sickling is a reversible phenomenon. Withincreased oxygen tensions, sickled cells depolymerize. This process ofpolymerization-depolymerization is very damaging to red cell membranesand eventually leads to irreversibly sickled cells (ISC) which retaintheir abnormal shape even when fully oxygenated. The average ISCsurvives for about 20 days in the body, as compared to the normal 120day life span. Individuals with HbS syndromes have, frequent infections,chronic hemolysis with a striking reticulocytosis andhyperbilirubinemia. The course of the disease is typically punctuatedwith a variety of painful crises called vaso-occlusive crises. Thesecrises represent episodes of hypoxic injury and infarction in theorgans, abdomen, chest, extremities or joints. Leg ulcers are anadditional manifestation of the vaso-occlusive tendency of this disease.Central nervous system involvement is common producing seizures and evenstrokes. Aplastic crises, also common, represent a temporary cessationof bone marrow activity and may be triggered by infections, folic aciddeficiency or both. Crises are episodic and reversible, but may befatal. Damage from crisis episodes tends to be cumulative and even inthose individuals with milder forms of sickle cell disorders, life-spanscan be greatly reduced. Absent alternative intervention, patientstypically die before the age of 30.

[0014] The thalassemia syndromes are a heterogeneous group of disordersall characterized by a lack of or a decreased synthesis of the globinchains of HbA. Deficiencies of β-globin expression are referred to asβ-thalassemias and deficiencies of α-globin, α-thalassemias. Thehemolytic consequences of deficient globin chain synthesis result fromdecreased synthesis of one chain and also an excess of the complementarychain. Free chains tend to aggregate into insoluble inclusions withinerythrocytes causing premature destruction of maturing erythrocytes andtheir precursors, ineffective erythropoiesis, and the hemolysis ofmature red blood cells. The underlying defects of hemoglobin synthesishave been elucidated over the years and largely reside in the nucleicacid sequences which express or control the expression of α or β globinprotein.

[0015] Surprisingly, α-thalassemias tend to be less severe than βthalassemias. Homozygous pairs of β chains are believed to be moresoluble than those derived from unpaired a chains. Consequently, theeffects associated with free or improperly paired globin chains, whichcorrelate with at least half of the clinical pathology associated withthalassemia, are minimized.

[0016] Hemoglobin H disease, a more severe form of a thalassemia, is adeletion of three of the four α globin genes. It is rarely found inthose of African origin, but mostly in Asians. With only a single αgene, α chain expression is markedly depressed and there is an excess ofβ chains forming tetramers called HbH hemoglobin. HbH is unable towithstand oxidative stress and precipitates with vessels or is removedby the spleen. The most severe form of a thalassemia is hydrops fetalisand results from a deletion of all α globin genes. In the fetus,tetramers of γ globin develop (Hb Barts) that have an extremely highoxygen lafty and are unable to release oxygen to the tissues. Severetissue anoxia results and leads to intrauterine fetal death.

[0017] Fetal β-type globin, or γ globin, is expressed in the earlieststages of mammalian development and persists until about 32 to 34 weeksof gestation. At this stage, the adult forms of β globin begin to beexpressed and substitute for the fetal proteins. Studies correlatingclinical hematological results with the locations of various mutationsthat correspond to switching indicate that a region located upstream ofthe 5′-end of the 3-gene may be involved in the cis suppression ofγ-gene expression in adults (E. F. Fritsch et al., Nature 279:598-603,1979). The reason for this switch from fetal to adult protein is unknownand does not appear to provide any significant benefit to the adult.

[0018] Each β globin gene comprises three exons which encode about 146amino acids, two introns and a 5′-untranslated region containing thepromoter sequences. Biosynthesis of β globin begins with transcriptionof the entire gene followed with RNA processing of the message, removalof the introns by splicing, poly A addition, capping andpost-transcriptional modifications. The mature mRNA molecule is exportedfrom the nucleus and translated into β globin. Defects in each of thesefunctions have been found associated with specific thalassemias.Identified mutations include single-nucleotide deletions, insertions andsubstitutions, frame shift mutations, deletions of entire segments ofcoding or controlling regions, improper termination signals, aberrantsplicing signals, and multiple mutations. β°-thalassemias arecharacterized by a complete absence of any β globin chains.β⁺-thalassemias are characterized by a detectable presence of a reducedamount of β chains.

[0019] There are three principal categories of β-thalassemia,thalassemia major, thalassemia intermedia and thalassemia minor.Patients with thalassemia minor may be totally asymptomatic and aregenotypically β⁺/β or β°/β. Although red cell abnormalities can bedetected, symptoms are mild. Thalassemia intermedia patients are mostoften genotypically β⁺/β⁺ or β°/β and present severe symptoms which canbe alleviated with infrequent blood transfusions. In contrast,thalassemia major patients are genotypically β°/β°, β°/β⁺ or β⁺/β⁺, andrequire regular and frequent transfusions. Children suffer from severegrowth retardation and die at an early age from the profound effects ofanemia. Those that survive longer suffer from morphological changes. Theface becomes distorted due to expansion of marrow within the bones ofthe skull, hepatosplenomegaly ensues, there is a delayed development ofthe endocrine organs including the sexual organs, and a progressive ironoverload with secondary hemochromatosis.

[0020] There are two direct consequences of β-thalassemia. First, thereis an inadequate formation of HbA and, therefore, an impaired ability totransport oxygen. There are also multiple effects attributable to animbalance between a and β chain synthesis. Surprisingly, thepathological consequences of globin chain imbalance appears to be themore severe. Free α chains form unstable aggregates that precipitatewithin red cell precursors in the form of insoluble inclusions. Theseinclusions damage cellular membranes resulting in a loss of potassium.The cumulative effect of these inclusions on the red blood cells is anineffective eropoiesis. An estimated 70% to 85% of normoblasts in themarrow are eventually destroyed. Those that do escape immediatedestruction are at increased risk of elimination by the spleen wheremacrophages remove abnormal cells. Further, hemolysis triggers anincreased expression of erythropoietin which expands populations oferythroid precursors within bone marrow and leads to skeletalabnormalities. Another severe complication of β thalassemia is thatpatients tend to have an increased ability to absorb dietary iron. Asmost treatments for thalassemia involve multiple transfusions of redblood cells, patients often have a severe state of iron overloaddamaging all of the organs and particularly the liver. To reduce theamount of iron in their systems, iron chelators are typicallyadministered. Although helpful, patients succumb at an average ofbetween about 17 to 35 years of age to the cumulative effects of thedisease and iron overload.

[0021] Genotypic variation in healthy individuals have been identifiedwherein adult β globin is not formed, but severe complications areavoided. These patients constituitively express fetal or γ globinprotein in amounts sufficient to substitute for the missing β globinprotein. This hereditary persistence of fetal hemoglobin (HPFH) mayinvolve one or both of the fetal β-globin genes, A-γ and G-γ.Apparently, consistent production of either γ-globin proteinaccomplishes the necessary functions, at least in the short term, of theabnormal or missing β-globin protein (R. Bernards et al., Nuc. AcidsRes. 8:1521-34, 1980).

[0022] A variety of small molecules have been shown to effect hemoglobinor fetal globin expression. Early experiments demonstrated that acetate(CH₃COOH), propionate (CH₃CH₂COOH), butyrate (CH₃CH₂CH₂COOH) andisobutyrate (CH₃CH(CH₃)COOH) all induced hemoglobin synthesis incultured Friend leukemia cells (E. Takahashi et al., Gann 66:577-80,1977). Additional studies showed that polar compounds, such as acidamides, and fatty acids could stimulate the expression of both fetal andadult globin genes in murine erythroleukemia cells (U. Nudel et al.,Proc. Natl. Acad. Sci. USA 74:1100-4, 1977). Hydroxrurea (H₂NCONHOH),another relatively small molecule, was found to stimulate globinexpression (N. L. Letvin et al., N. Engl. J. Med. 310:869-73, 1984).Stimulation, however, did not appear to be very specific to fetal globin(S. Charache et al., Blood 69:109-16, 1987). Hydroxyurea is also awell-known carcinogen making its widespread and long term use as apharmaceutical impractical.

[0023] Expression from the γ-globin genes has been successfullymanipulated in vivo and in vitro using agents such as cytosinearabinoside (AraC), a cytotoxic agent that induces fetal reticulocyteproduction (P. Constantoulakis et al., Blood 74:1963-71, 1989), and5-azacytidine (AZA), a well-known DNA methylase inhibitor (T. J. Ley etal., N. Engl. J. Med. 307:1469-75, 1982). Continuous intravenousadministration of AZA produced a five- to seven-fold increase in γglobin mRNA of bone marrow cells (T. J. Ley et al., Blood 62:370-380,1983). Additional studies have shown that there are significantalterations in the population of stem cells in the bone marrow after AZAtreatment (A. T. Torrealba-De Ron et al., Blood 63:201-10, 1984). Theseexperiments indicate that AZA's effects may be more attributable toreprogramming and recruitment of erythroid progenitor cells than to anydirect effects on specific gene expression. Many of these agentsincluding AZA, AraC and hydroxyurea are myelotoxdc, carcinogenic orteratogenic making long-term use impractical.

[0024] One of the major breakthroughs in the treatment ofhemoglobinopathies was made when it was discovered that butyric acid(butanoic acid; CH₃CH₂CH₂COOH) accurately and specifically stimulatedtranscription of the human fetal (γ) globin gene (G. A. Partington etal., EMBO J. 3:2787-92, 1984). These findings were quickly confirmed invivo wherein it was shown that pharmacological doses of butyric acidgreatly increased expression of fetal globin in adult chickens renderedanemic by injections with phenylhydrazine (G. D. Ginder et al., Proc.Natl. Acad. Sci. USA 81:3954-58, 1984). Selective transcriptionalactivation was again thought to be due to hypo-methylation of theembryonic gene (L. J. Burns et al., Blood 72:1536-42, 1988). Othersspeculated that histone acetylation, a known effect of butric acid, maybe at least partly responsible for increased fetal gene expression (L.J. Burns et al., EMBO J. 3:2787, 1984).

[0025] Over 50 derivatives of butyric acid have since been found to beeffective in stimulating fetal globin production (S. P. Perrine et al.,Biochem. Biophys. Res. Commun. 148:694-700, 1987). Some of these includebutric acid salts such as sodium and arginine butyrate, α-amino-n-butyncacid (butyramide; CH₃CH₂CH₂CONH₂), and isobutyramide (CH₃CH(CH₃)C0NH₂).Although promising in pilot clinical studies, treated patients wereunable to maintain adequate levels of fetal globin in their system. Itwas later determined that many of these forms of butyric acid hadextremely short-half lives. Oxidation in the serum, clearance byhepatocytes and filtration through the kidneys rapidly eliminated theseagents from the patient's system. With others, patients rapidlydeveloped tolerance or metabolites of compounds had the opposite desiredeffect.

[0026] A number of aliphatic carboxylic acids have been tested for theirability to specifically increase fetal globin expression in K562 humanerythroleukernia cells (S. Safaya et al., Blood 84:3929-35, 1994).Although longer chains were considered toxic to cells, propionate(CH₃CH₂COOH) and valerate (pentatonic acid; CH₃CH₂CH₂CH₂COOH) were foundto be most effective. Butyrate (CH₃(CH₂)₂COOH), caproate(CH₃(CH₂)₄COOH), caprylate (CH₃(CH₂)₆COOH), nonanoate (CH₃(CH₂)₇COOH),and caprate (CH₃(CH₂)₈COOH) produced much less of an effect. Phenylacetate (C₆H₅CH₂COOH) and its precursor, 4-phenyl butyrate(C₆H₅CH₂CH₂CH₂COOH), were found to decrease fetal globin expressingreticulocyte proliferation, but increase relative proportions of fetalglobin per cell in cultured erythroid progenitor cells (E. Fibach etal., Blood 82:2203-9, 1993). Acetate (CH₃COOH), a metabolic product ofbutyrate catabolism, increased both erythrocyte precursor populationsand also fetal globin synthesis. However, these studies alsodemonstrated that positive effects could only be maintained for veryshort periods of time (B. Pace et al., Blood 84:3198-204, 1994).

[0027] Other agents shown to affect fetal globin expression includeactivin and inhibin. Inhibin, a disulfide linked hormone of twosubunits, suppresses secretion of follicle-stimulating hormone from thepituitary gland. Activin, sometimes referred to as erythroiddifferentiating factor (EDF) or follicle-stimulating hormone releasingprotein (FRP), is also a hormone and both of these macromoleculesinduced hemoglobin accumulation in cultured human erythrocytes (S. P.Perrine et al., Blood 74:114a, 1989). Recently, studies have shown thatsteel factor, a product of the mouse steel locus (D. M. Anderson et al.,Cell 63:235-43, 1990), is also capable of influencing fetal globinsynthesis in erythroid progenitors (B. A. Miller et al., Blood79:1861-68, 1992).

[0028] Other methods to increase fetal globin expression have focused onrecruitment and reprogramming of erythroid progenitor cells to increasetotal globin expression. For example, the hematopoietic growth factorerythropoietin (EPO) was found to be a potent, although not afetal-specific, reticulocyte stimulator (Al-Khatti et al., Trans. Assoc.Am. Physicians 101:54, 1988; G. P. Rodgers et al., N. Engl. J. Med.328:73-80, 1993). In one experiment, animals were treated with EPOfollowing a specific course of therapy (U.S. Pat. No. 4,965,251).According to this experiment, a high dose of eiythropoietin wasadministered in a first time period followed by a second time periodwherein erythropoietin was withheld. Following this regimen oftreatment, typical for a cytokine, F-reticulocyte obtained from twochronically -anemic baboons increased from 6-8% and 20% pre-treatment to23% and 50% post-treatment, respectively.

[0029] These methods were somewhat advantageous to artificiallyphlebotomized baboons, but could be counter-productive to patients witha hemoglobinopathy. Thalassemic patients express high levels of EPO,supplemental treatments with EPO and do not improve the globin chainimbalance, but result in more thalassemic cells. Sickle cell patientsand other patients with unstimulated levels would also not benefit fromsupplemental EPO treatments because absolute amounts of both α-globinand non α-globin would increase. Treatments with EPO can increase thefrequency and number of sickle cell crises due to increasing the bloodviscosity with more Hbs, both of which are to be avoided in suchpatients.

[0030] Other hematopoietic growth factors, such asgranulocyte/macrophage-colony stimulating factor (GM-CSF) andinterleukin 3 (IL-3), were also tested in vivo or in vitro for theability to stimulate F-reticulocytes (M. Giabbianell et al., Blood74:2657, 1989; A. R. Migliaccio et al., Blood 76:1150, 1990). Both ofthese factors were found to non-specifically increase fetal-globinsynthesis in tissue culture cells.

SUMMARY OF THE INVENTION

[0031] The invention provides novel compositions and methods for thetreatment and prevention of blood disorders.

[0032] We have found that certain compositions provide improvedadvantages such as prolonged induction of growth related genes, e.g.,C-myb and C-myc gene, unexpectedly better cell proliferation, enhancedstability, and have a sparing or abrogating effect for growth factorrequirements such as IL-3 or EPO.

[0033] The compositions include C₁-C₄ alkyl and/or phenyl substitutionon carboxylic acids such as α-methylhydrocinnamic acid, 3,4dimethoxycinnamic acid, 2-methylhydrocinnamic acid, 2- and3-methoxycinnamic acid, 3,4 dimethoxyphenylacetic acid, 3-3,4dimethoxyphenylpropionic acid, 2,5 dimethoxyphenylacetic acid, 2,2dimethylbutyric acid, 2,2 dimethylpropionic acid, 2,2dimethylphenoxyacetic acid, 2,2 dimethymethoxyacetic acid, and 2,2dimethylphenylpropionic acid. The alkyl group can be substituted ornon-substituted. Substituents include hydroxy, halogens phenyl, thiol,mercapto, and methylthiol, Dimethyl substitutions onto the carboxylicacids are preferred. Pharmaceutically acceptable salts of thesecompositions are also included herein.

[0034] The compositions can be administered by any of a range ofmethods. Preferred methods include as oral compositions or by pulseadministration.

[0035] One embodiment of the invention is directed to methods for thetreatment of blood disorders and other maladies such as neoplasia byadministering compositions to a patient in pulses. Pulse therapyaccording to the methods of the invention is much more effective thancontinuous therapy. The effective dose as well as the total amount ofcomposition needed by the patient to be therapeutically effective isdecreased as compared to amounts required for similar effect withcontinuous therapy. Further, as most chemical compositions are non-toxicat all effective doses, pulsed administration can be continued for verylong periods with no adverse effects to the patient.

[0036] Another embodiment of the invention is directed to methods forthe stimulation of cell proliferation by the administration oferythropoietin or other cell stimulatory agent to a patient and theadministration of a chemical composition of the invention in pulses.Such a treatment regimen prepares bone marrow cells for stimulation andincreases overall hemoglobin expression and production in the body.

[0037] These compositions can be used, either with or without pulsing,for the treatment of not only blood disorders, but for other disorderssuch as neoplasia.

[0038] Other objects and advantages of the invention are set forth inpart in the description which follows, and in part, will be obvious fromthis description, or may be learned from the practice of the invention.

DESCRIPTION OF THE DRAWINGS

[0039]FIG. 1 shows primer extension analysis of globin mRNA demonstratesa 2.4-26 fold increase in γ-globin mRNA was induced over constitutivelevels in untreated control K562 cells by Arg (arginine butyrate), PAA(ST 1; phenoxyacetic acid), ST 7 (AMHCA; α-methylhydrocinnamic acid), ST20 (DMB or DMBA; 2,2 dimethylbutyric acid), ST 32 (2-methoxycinnamicacid), ST 33 (2 methyl hydrocinnamic acid), ST 34 (cis-2-methoxycinnamicacid), ST 37 (3,4 dimethoxy phenyl acetic acid), ST 38 (3-3,4-dimethoxyphenyl propionic acid), ST 40 (2,5 {dimethoxy phenyl} acetic acid), ST44 (3,5 dimethoxy 4-hydroxy cinnamic acid), ST 47 (transcinnamic acid).Butyric acid produced a 2-fold increase in γ-globin expression comparedto untreated control cells. Fold increase over control levels is shown.

[0040]FIGS. 2A and 2B show comparisons on cell proliferation. FIG. 2Ashows comparison of the proliferation of 32D cells in the presence ofoptimal IL-3 (25 U/ml), low IL-3 (0.5 U/ml; 50 fold depletion) and inthe absence of IL-3, which results in uniform cell death by apoptosis.

[0041]FIG. 2B shows comparison of proliferative rates of multi-lineageIL-3 dependent cells in the presence of a low concentration of IL3 aloneand with the addition of erythropoietin (EPO) at 3 U/ml. G-CSF(granulocyte-colony stimulating factor) at 100 U/ml, and 1.0 mMconcentrations of PAA, AMHCA, DMB PMBA), butyric acid, DMHAA(dimethylhydroxyacetic acid). Withdrawal of IL-3 completely and additionof butyrate to the low concentration (0.5 U/ml) of IL-3 resulted indecreased cell proliferation and cell death. Addition of test compoundsresulted in continued cell proliferation at rates similar to thoseinduced by EPO and G-CSF.

[0042]FIG. 3 shows induction of reticulocytes in C57 mice treated withAMHCA (ST 7 or ST 007) for 7 days. Increases of 2.5 and 6 fold overbaseline reticulocytes was observed (shows a 3 and 6 fold increase inRBC production). The treatment period is shown by the horizontal barabove the graph. A similar increase was not observed in controls whichwere similarly handled and treated with saline and bled (phlebotomized)the same amount for 21 days. Controls had no significant increase inredculocyte counts.

[0043]FIG. 4 shows pharmacokinetics after oral administration of singledoses of PAA, DMBA (in humans) and AMHCA (in monkeys) in primates.Plasma levels persisted in the millimolar range far above concentrationswhich are necessary for hematologic effects in vitro (shown by arrow)for greater than 6 hours following oral doses of 40-500 mg/kg bodyweight. This demonstrates that these compounds are useful in vivo andare resistant to rapid metabolism.

[0044]FIG. 5 shows the rate of increase in c-myb and c-myc expression in32D cells compared to control cells cultured with low IL-3 treated withG-CSF (positive control), EPO (positive control), ST 7 or 7 (AMHCA), ST14 or 14 (DMHCA; 2, 2 dimethylhydrocinnamic acid), ST 20 or 20 (DMBA),PAA, ST 30 or 30 (BMHCA or β-aminohydrocinnamic acid), DL-βABA (DL-βamino butyric acid), ST 24 or 24 (DMPA; 2,2 dimethyl propionic acid),and ST 27 or 27 (DMMAA or 2, 2 dimethyl methoxy acetic acid). White barsrepresent fold increases at day 1 and black bars fold increases at day7. Baseline (or the 0 level) represents 0.5 U/ml IL-3. The myb gene hasbeen shown to be an important regulator of hematopoietic cellproliferation, differentiation and apoptosis.

[0045]FIG. 6 shows the rate of increase or decrease in histone and actinexpression (as negative control) in 32D cells treated with G-CSF, EPO,ST 7, 14, 20, PAA, 30, DL-βABA, 24 and 27. No significant change in theexpression of these genes was observed with exposure to the testcompounds. This demonstrates that the increase in c-myb and c-mycexpression is specific.

[0046]FIG. 7 shows the rate of proliferation of 32D cells with low IL-3(0.5 U/ml) after treatment with AMHCA (ST 007) (to increase c-myc andc-myb expression) as compared to treatment with butyrate. Cells die andare do not proliferate in the presence of butyrate whereas proliferationincreases 5 fold over 4 days in the presence of ST 007 (i. e. increasedc-myc and c-myb expression translates into increased cellularproliferation).

[0047]FIG. 8 shows hematologic effects of ST 007 in a Baboon (RBCproliferation translates from in vitro data to in vivo data).

[0048]FIG. 9 shows effects of ST 7 on hemoglobin and platelets (i. e. ST7 acts on multiple cell lineages).

[0049]FIG. 10 shows how compounds act on very primitive andmultipotential stem cells as shown in this chart.

[0050]FIG. 11 shows Northern Blots for the growth related genes.

[0051]FIG. 12 shows the increase in reticulocytes vs. days of treatmentin mice with phenylacetic acid.

[0052]FIG. 13 shows white blood cell stimulation in a baboon by amethylhydrocinnamic acid (AMHCA).

DESCRIPTION OF THE INVENTION

[0053] As embodied and broadly described herein, the present inventionis directed to compositions and methods for the administration ofpharmaceutical compositions useful for the treatment and prevention ofdisorders including cell proliferative disorders such as malignanciesand cytopenias, and blood disorders such as an anemia, sickle cellsyndrome and thalassemia.

[0054] We have found that a number of compositions provide excellentresults in treating many of these disorders. The compounds includeα-methylhydrocinnamic acid (trans and cis); 2-methylhydrocinnamic acid(trans and cis); 2- and 3-methoxycinnamic acid (trans and cis);4-chlorophenoxy -2-propionic acid; 3,4 dimethoxycinnamic acid; 3,4dimethoxyphenyl acetic acid; 3-3,4 dimethoxy phenyl propionic acid;2-(4′-methoxyphenoxy)propionic acid; 2,5 dimethoxyphenyl acetic acid;hydrocinnamic acid; 3-phenylpropionic acid; 2,2 dihydrocinnamic acid;2,methylbutyric acid, 2,2 dimethylbutyric acid; 2,2 dimethylpropionicacid; 2,2 dimethylphenoxy acetic acid; 2,2 dimethylmethoxy acetic acid;2,2 dimethylphenyl propionic acid; α-methyl lactate methyl ether;benzoyl formic acid; D,L α-amino butyric acid; D,L 0-amino butyric acid;β-aminohydrocinnamic acid; α-methyl lactic acid; and dimethylhydroxyacetic acid.

[0055] A preferred group of compositions include C₁-C₄ substitutedand/or phenyl-substitutions on carboxylic acids. Preferably it is aC₁-C₄ alkyl substitution. The alkyl or phenyl moiety can be substitutedor non-substituted. Preferred substituents include hydroxy, halogens,phenyl, thiol, mercapto and methyl thiol.

[0056] Preferred carboxylic acids include cinnamic acids (such ashydrocinnamic acid), acetic acids and propionic acids.

[0057] The C₁-C₄ alkyl is preferably methyl. Preferably, it is adimethyl substitution.

[0058] Preferred compounds include C₁-C₄ substituted phenoxyacetic acid,C₁-C₄ substituted cinnamic acid, C₁-C₄ phenoxy acetic acid, C₁-C₄substituted propionic acid and C₁-C₄ substituted butyric add. Morepreferred compounds include C₁-C₄ alkyl and/or phenyl substitution oncarboxylic adds such as α-methylhydrocinnamic acid, 3,4dimethoxycinnamic acid, 2-methylhydrocinnamic acid, 2- and3-methoxycinnamic acid, 3,4 dimethoxyphenylacetic acid, 3-3,4dimethoxyphenylpropionic acid, 2,5 dimethoxyphenylacetic acid, 2,2dimethylbutyric acid, 2,2 dimethylpropionic acid, 2,2dimethylphenoxyacetic acid, 2,2 dimethymethoxyacetic acid, and 2,2dimethylphenylpropionic acid. The alkyl group can be substituted ornon-substituted. Substituents include hydroxy, halogens phenyl, thiol,mercapto, and methylthiol, Dimethyl substitutions onto the carboxylicacids are preferred. Pharmaceutically acceptable salts of thesecompositions are also included herein.

[0059] These compounds can be administered by known techniques such asorally, intraperitoneally, etc.

[0060] Preferably, the compounds are manufactured in such means thatthey can be administered orally.

[0061] In another embodiment, the compounds are administeredintravenously.

[0062] In a preferred method they are delivered by pulse therapy.

[0063] It has been discovered that a variety of chemicals useful for thetreatment of blood and other disorders are more effective whenadministered to a patient in pulses. Pulse therapy is not a form ofdiscontinuous administration of the same amount of a composition overtime, but comprises administration of the same dose of the compositionat a reduced frequency or administration of reduced doses.

[0064] One embodiment of the invention is directed to compositions witha mechanism of action involving regulation of histone deacetylase by achemical compound such as glycerol, acetic acid, butric acid, and anamino-n-butric acid (such as d- or 1-amino-n-butyric acid, α- orβ-amino-n-butyric acid). Some butyric acid compounds, such as argininebutyrate or isobutamide may also be useful. See also, U.S. Pat. Nos.4,822,821 and 5,025,029. Thus, one can regulate histone deacetylase toenhance globin production by administering an effective amount of acompound selected from the group consisting of glycerol, acetic acid,butyric acid, and amino-n-butyric acid, in a pharmaceutically acceptablecarrier or diluent. Preferably, the compound is an amino-n-butric acid.

[0065] According to these methods, blood and other disorders can beeffectively treated and without unnecessary adverse side effects to thepatient. Although most compositions are generally safe and non-toxic attherapeutic doses, pulsed administration further reduces risksassociated with, for example, toxicity, allergic reactions, the build-upof toxic metabolites and inconveniences associated with conventionaltreatment. In addition, chemical compositions, being useful at a reduceddose and frequency, have a substantially reduced risk of inducedtolerance. Drugs are not inactivated by cellular enzymes or cleared fromcells and organs prior to having the desired effect. Further, long-termtherapy, typically required for the amelioration of many blooddisorders, becomes possible. Consequently, doses necessary formaintaining a constant effect for the patient are steady and materialcosts and inconveniences associated with administration aresubstantially reduced.

[0066] One embodiment of the invention is directed to the pulsedadministration of pharmaceutical compositions for the treatment orprevention of a blood disorder. Pulsed administration is surprisinglymore effective than continuous treatment as pulsed doses are often lowerthan would be expected from continuous administration of the samecomposition. Each pulse dose can be reduced and the total amount of drugadministered over the course of treatment to the patient is minimized.

[0067] In traditional forms of therapy, repeated administration isdesigned to maintain a desired level of an active ingredient in thebody. Very often, complications that develop can be attributed to dosagelevels that, to be effective, are near toxic or otherwise harmful tonormal cells. In contrast, with pulse therapy, in vivo levels of drugdrop below that level required for effective continuous treatment.Therefore, pulsing is not simply the administration of a sufficientlylarge bolus such that there will be therapeutically sufficient drugavailable for a long period of time. Pulsed administration cansubstantially reduce the amount of the composition administered to thepatient per dose or per total treatment regimen with an increasedeffectiveness. This represents a significant saving in time, effort andexpense and, more importantly, a lower effective dose substantiallylessens the number and severity of complications that may be experiencedby the patients. As such, pulsing is surprisingly more effective thancontinuous administration of the same composition.

[0068] Preferably, compositions contain chemicals that are substantiallynon-toxic. Substantially non-toxic means that the composition, althoughpossibly possessing some degree of toxicity, is not harmful to thelong-term health of the patient. Although the active component of thecomposition may not be toxic at required levels, there may also beproblems associated with administering the necessary volume or amount ofthe final form of the composition to the patient. For example, if thecomposition contains a salt, although the active ingredient may be at aconcentration that is safe and effective, there can be a harmfulbuild-up of sodium, potassium or another ion. With a reduced requirementfor the composition or at least the active component of thatcomposition, the likelihood of such problems can be reduced or eveneliminated. Consequently, although patients may have minor or short termdetrimental side-effects, the advantages of taking the compositionoutweigh the negative consequences.

[0069] Methods for the pulsed administration of compositions of theinvention are preferably used for the treatment of blood disorders suchas hemoglobinopathies (e.g. sickle cell anemia, thalassemia), neoplasticdiseases including tumors, leukemias, lymphoproliferative disorders andmetastases, and cell proliferative disorders such as viral-inducedmalignancies (e.g. latent virus infections) and cytopenia including redand white blood cell anemia, leukopenia, neutropenia andthrombocytopenia. Compositions most effective at pulsed administrationare typically non-toxic or non-cytotoxic chemicals without anysubstantial proteinaceous active component at the therapeuticallyeffective pulsed dose. Preferably, treatment does not stimulateapoptosis in the cells being directly treated or in the otherwise normalcells of the body which will also be exposed to the composition.

[0070] Individual pulses can be delivered to the patient continuouslyover a period of several hours, such as about 2, 4, 6, 8, 10, 12, 14 or16 hours, or several days, such as 2, 3, 4, 5, 6, or 7 days, preferablyfrom about 1 hour to about 24 hours and more preferably from about 3hours to about 9 hours. Alternatively, periodic doses can beadministered in a single bolus or a small number of injections of thecomposition over a short period of time, typically less than 1 or 2hours. For example, arginine butyrate has been administered over aperiod of 4 days with infusions for about 8 hours per day or overnight,followed by a period of 7 days of no treatment. This has been shown tobe an effective regimen for many thalassemic disorders. Fetal hemoglobinlevels rise substantially and there is a significant rise in the numberof both adult and fetal hemoglobin expressing cells. Substantially meansthat there are positive consequences that raise the patient's standardof living such as, for example, increased activity or mobility, fewerside-effects, fewer hospital stays or visits to the physician, or fewertransfusions.

[0071] The interval between pulses or the interval of no delivery isgreater than 24 hours and preferably greater than 48 hours, and can befor even longer such as for 3, 4, 5, 6, 7, 8, 9 or 10 days, two, threeor four weeks or even longer. As the results achieved may be surprising,the interval between pulses, when necessary, can be determined by one ofordinary skill in the art. Often, the interval between pulses can becalculated by administering another dose of the composition when thecomposition or the active component of the composition is no longerdetectable in the patient prior to delivery of the next pulse. Intervalscan also be calculated from the in vivo half-life of the composition.Intervals may be calculated as greater than the in vivo half-life, or 2,3, 4, 5 and even 10 times greater the composition half-life. Forcompositions with fairly rapid half lives such as arginine butyrate witha half-life of 15 minutes, intervals may be 25, 50, 100, 150, 200, 250300 and even 500 times the half life of the chemical composition.

[0072] The number of pulses in a single therapeutic regimen may be aslittle as two, but is typically from about 5 to 10, 10 to 20, 15 to 30or more. In fact, patients can receive drugs for life according to themethods of this invention without the problems and inconveniencesassociated with current therapies. Compositions can be administered bymost any means, but are preferable delivered to the patient orally or asan injection (e.g. intravenous, subcutaneous, intraarterial, infusion orinstillation, and more preferably by oral ingestion. Various methods andapparatus for pulsing compositions by infusion or other forms ofdelivery to the patient are disclosed in U.S. Pat. Nos. 4,747,825;4,723,958; 4,948,592; 4,965,251 and 5,403,590.

[0073] Compositions administered in pulses have the surprising benefitof reducing the overall load of drug on the patient as the total amountof drug administered can be substantially less than that amount that hasbeen therapeutically administered by conventional continuous therapy.For example, arginine butyrate has been shown to be effective atcontinuous administration at about 2000 mg/kg patient weight. Doses ofbetween about 400 to 1500 mg/kg, preferably from about 600 to 1000 mg/kgand more preferably from 700 to 800 mg/kg, when administered in pulses,are surprisingly more beneficial as measured by a rise in fetalhemoglobin levels in thalassemic patients. Typical pulsed amounts ofarginine butyrate are from about 2 to about 20 g/kg/month, andpreferably from about 3 to about 10 g/kg/month wherein the patientreceives a total of less than about 20 kg per month, preferably lessthan about 15 kg per month and more preferably less than about 10 kg permonth. The amounts administered per pulse as well as the total amount ofthe composition received by the patient over the regimen issubstantially reduced. Preferably, the therapeutically effective pulseddose is less than the continuous dose, or less than one half, one third,one quarter, one fifth, one tenth or even one twentieth of thetherapeutic continuous dose of the same composition or even less.

[0074] A treatment regimen can be considered effective if it stimulatesglobin chain expression or the proliferation of erythroblasts or othererythroid progenitor cells, for example with hemoglobinopathy patients,the proliferation of cells such as white blood cells or platelet formingcells, or reduces the number of proliferating cells in, for example, atumor or other malignancy. Cell numbers are usually most easilydetermined from peripheral blood sampling or from calculations of tumorsize.

[0075] Another embodiment of the invention is directed to methods forthe pulsed administration of compositions to a patient along with thepulsed or non-pulsed administration of other compositions or therapiesfor the treatment or amelioration of a disorder. Pulsing of either orboth of the compositions can, in part, synchronize cell development, asthere is an increased proliferation of erythrocytes and an increasedexpression of hemoglobin, specifically, fetal hemoglobin. Compositionsand therapies which can be pulsed include most of the known orconventional or already well-known treatment regimens. One preferabletreatment involves the pulsed or continuous administration oferythropoietin, or another bone marrow cell stimulant, followed by thepulsed administration of a chemical composition of the invention. Thisregimen has the beneficial effect of stimulating the process of E/Megacell to erythrocyte development and proliferation which can be followedby stimulation of fetal globin gene expression from the newlyproliferated cells. Following such treatments, fetal globin levels inthe body rise substantially and much higher than would have beenexpected from conventional continuous therapy.

[0076] A blood disorder is any disease or malady which could becharacterized as a direct or indirect consequence of a defect or diseaseof hemoglobin producing cells or the production of hemoglobin. The blooddisorder may be associated with an anemia such as sickle cell anemia,hemolytic anemia, infectious anemia, aplastic anemias, hypoproliferativeor hypoplastic anemias, sideroblastic anemias, myelophthisic anemias,antibody-mediated anemias, anemias due to enzyme-deficiencies or chronicdiseases, anemias due to blood loss, radiation therapy or chemotherapy,thalassemias including α-like and β-like thalassemias. Treatable blooddisorders also include syndromes such as hemoglobin C, D and E disease,hemoglobin lepore disease, and HbH and HbS diseases. Treatmentameliorates one or more symptoms associated with the disorder. Symptomstypically associated with blood disorders include, for example, anemia,tissue hypoxia, organ dysfunction, abnormal hematocrit values,ineffective erythropoiesis, abnormal reticulocyte (erythrocyte) count,abnormal iron load, the presence of ring sideroblasts, splenomegaly,hepatomegaly, impaired peripheral blood flow, dyspnea, increasedhemolysis, jaundice, anemic crises and pain such as angina pectoris.

[0077] Compositions to be administered according to the methods of theinvention are preferably physiologically stable and safe, and containone or more chemical compounds that increase the extent or magnitude ofhematopoiesis, increase the proliferation of hemoglobin expressing andother cells, increase or balance the expression of globin proteins orincrease or stimulate the specific expression of functional globinprotein such as γ-globin. Stimulation of specific gene expressioninvolves activation of transcription or translation promoters orenhancers, or alteration of the methylation pattern or histonedistribution along the gene to promote expression. Expression may alsobe stimulated by inhibition of specific transcription or translationrepressors, activation of specific transcription or translationactivation factors, or activation of receptors on the surface ofparticular populations of cells. Stimulation may recruit additionalcells to marrow, reprogram differentiated cells to express hemoglobin orswitch to the expression of an embryonic, fetal or other globin-likepeptide. Stimulation may also activate a previously dormant orrelatively inactive genes which substitutes for the defective or damagedgene products such as, for example, the post-natally suppressed geneswhich encode ε, δ or γ globin, which can substitute for adult β globin,or ζ globin which can substitute for a defective or deficient α globin.

[0078] Alternatively, compositions may be used to turn down theexpression of those genes whose products are being over expressed andthereby disrupting the balanced production of normal globin proteins.Genes whose expression or whose balanced expression can be effected bythe compositions include the globin genes such as the various forms ofthe ζ-type genes, the ε-type genes, the α-type genes, the β-type genes,the δ-type genes, the γ-type genes and at least partially functionalpseudo-globin genes.

[0079] The mechanism of action of many of the chemical compounds oractive ingredients of compositions for the treatment of blood disordersinvolves effecting one or more of the processes of cell proliferation,cell recruitment, specific hemoglobin expression, heme synthesis orglobin chain synthesis. Cell proliferation may be increased, forexample, by stimulating stem cells, CFUs, BFUs, megakaryocytes, myeloidcells, platelets, white blood cells or pro-erythrocyte colony growth, ordecreased, for example, by effecting a cell's period in or ability totransverse a stage (S, G₀, G₁, M) of the cell cycle. Cell recruitmentmay be promoted through the expression of specific cytokines such ascell surface receptors or secreted factors. Hemoglobin expression can beincreased or decreased by affecting heme expression, globin peptideexpression, heme/globin peptide assembly, globin peptide glycosylationor globin transport through the golgi apparatus. Globin expression canbe increased or decreased by altering chromatin and/or nucleosomestructure to render α genetic element more or less susceptible totranscription, by altering DNA structure, for example, by methylation ofG residues, by affecting the activity of cell-specific transcription ortranslation factors such as activators or repressors, or by increasingthe rate of transcription or translation. For example, useful chemicalcompounds include C₁-C₄ alkyl substituted or phenyl substitutedcarboxylic acid compounds such as phenoxyacetic acid, methoxyaceticacid, substituted-cinnamic acid such as dimethyl hydrocinnamic acid,α-methyl cinnamic acid and α-methylhydrocinnamic acid (AMHCA) stimulatealterations in binding or removal of transcription factors from theproximal promoter region of certain genes of the γ- and β-globin geneclusters and thereby increase post-natally suppressed gene expression.

[0080] Chemical compounds preferably increase the expression ofhemoglobin, increase the expression of one or more embryonic or fetalglobin genes or increase the number of hemoglobin expressing or fetalglobin expressing reticulocytes. Preferably, compositions increaseembryonic or fetal globin gene expression or embryonic or fetalreticulocyte counts greater than about 2%, more preferably greater thanabout 5%, and even more preferably greater than about 9%. Forcomparative purposes, a 4% increase in fetal globin gene expressionequates to about 20% to 25% rise or increase in fetal globin inperipheral blood samples. Consequently, an increase of greater thanabout 1% fetal globin expression, preferably greater than about 3%, orabout 1% fetal globin expressing cells, preferably greater than about3%, can alleviate symptoms associated with beta globin disorders.

[0081] Hemoglobin expression, globin expression and cell proliferationcan be assayed by measuring fold increases in expressed amounts ofspecific protein or numbers of specific cells in treated samples ascompared to untreated controls. Using this criteria, compositionspreferably increase the amount of hemoglobin expression, the amount ofglobin expression, the number of hemoglobin expressing cells or thenumber of globin expressing cells by greater than or equal to abouttwo-fold, preferably about four-fold and more preferably abouteight-fold.

[0082] Chemical compounds are preferably optically pure with a specificconformation (plus {+} or minus {−}), absolute configuration (R or S),or relative configuration (D or L). Particular salts such as sodium,potassium, magnesium, calcium, choline, amino acid, ammonium or lithium,or combinations of salts may also be preferred, however, certain saltsmay be more advantageous than others. For example, chemical compoundsthat require high doses may introduce too much of a single salt to thepatient. Sodium is generally an undesirable salt because at high doses,sodium can increase fluid retention resulting in tissue destruction. Insuch instances, combinations of different salts or alternative salts canbe used.

[0083] In addition to the above chemical compounds, other compoundsinclude derivatives of these chemicals. Derivatives are chemical orbiological modifications of the parent compound and include analogs,homologs, next adjacent homologs and compounds based on any of theforegoing. Analogs include both structural and functional analogs.Functional analogs are those compounds which are functionally related tothe activity of the parent compound. Structural analogs are thosecompounds related to the parent compound in the arrangement or number ofcarbon atoms. For example, such compounds may have double or triplecovalent bonds wherein the parent has a single covalent bond. Homologsare those compounds which have the same number of carbon atoms as theparent compound, but further comprise additional moieties such as one ormore phosphate groups (PO₄), sulfate groups (SO₃), amines and amides(NH₃), nitrate groups (NO₂), acidified or esterified carbon atoms orcombinations thereof. Next adjacent homologs are those compounds withone more or less carbon atom. Related compounds include those compoundswhich have been modified such as by substitutions and/or additions. Forexample, compounds of the invention may be substituted with one or morehalogens such as chlorine (Cl), fluorine (F), iodine (I), bromine (Br)or combinations of these halogens. As known to those of ordinary skillin the art, halogenation can increase the polarity, hydrophilicity orlipophilicity or a chemical compound which can be a desirable feature,for example, to transform a chemical compound into a composition whichis more easily tolerated by the patient or more readily absorbed by theepithelial lining of the gastrointestinal tract. Such compositions couldbe orally administered to patients.

[0084] Therapeutically effective chemical compounds may be created bymodifying any of the above chemical compounds so that after introductioninto the patient, these compounds metabolize into active forms, such asthe forms above, which have the desired effect on the patient. Compoundsmay also be created which are metabolized in a timed-release fashionallowing for a minimal number of introductions which are efficacious forlonger periods of time. Combinations of chemical compounds can alsoproduce useful new compounds from the interaction of the combination.Such compounds may also produce a synergistic effect when used incombination with other known or other compounds.

[0085] Compositions may also comprise proteinaceous agents such ascytokines that will increase the extent or magnitude of hematopoiesis,increase the proliferation of hemoglobin expressing cells, increase orbalance the expression of hemoglobin macromolecules or increase orstimulate the specific expression of alternate globin genes such asγ-globin. Such proteinaceous agents include steel factor, insulin,erythropoietin (EPO), interferon (IFN), insulin growth factor (IGF),stem cell factor (SCF), macrophage-colony stimulating factor (M-CSF),granulocyte-colony stimulating factor (G-CSF), GM-CSF, growth factorssuch as fibroblast-derived growth factor (FGF), epidermal growth factor(EGF) and platelet-derived growth factor (PDGF), nerve growth factor(NGF), vascular endothelial growth factor (VEGF), bone morphogenicproteins (BMPs), the interleukins (IL) IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, etc., activin also referred to as erythroiddifferentiation factor (EDF) or follicle-stimulating hormone releasingprotein (FRP), inhibin, stem cell proliferation factor (SCPF) and activefragments, subunits, derivatives and combinations of these proteins.Erythropoietin, activin and SCF all stimulate the proliferation of stemcells, committed cells and erythroid progenitor cells, and can alsostimulate the expression of embryonic globin, fetal globin or partlyfunctional pseudo-globin expression. The hematopoietic factor, steelfactor, also referred to as kit ligand, mast cell growth factor and stemcell factor, recruits and stimulates the proliferation of hemoglobinexpressing cells and the specific expression of embryonic or fetalglobin. Proteinaceous agents of the invention may also be animated,glycosylated, acylated, neutralized, phosphorylated or otherwisederivatized to form compositions which are more suitable for the methodof administration to the patient or for increased stability duringshipping or storage.

[0086] Compositions may be physiologically stable at therapeuticallyeffective concentrations. Physiological stable compounds are compoundsthat do not break down or otherwise become ineffective upon introductionto a patient prior to having a desired effect. Compounds arestructurally resistant to catabolism, and thus, physiologically stable,or coupled by electrostatic or covalent bonds to specific reagents toincrease physiological stability. Such reagents include amino acids suchas arginine, glycine, alanine, asparagine, glutamine, histidine orlysine, nucleic acids including nucleosides or nucleotides, orsubstituents such as carbohydrates, saccharides and polysaccharides,lipids, fatty acids, proteins, or protein fragments. Useful couplingpartners include, for example, glycol such as polyethylene glycol,glucose, glycerol, glycerin and other related substances.

[0087] Physiological stability can be measured from a number ofparameters such as the half-life of the compound or the half-life ofactive metabolic products derived from the compound. Certain compoundsof the invention have in vivo half lives of greater than about fifteenminutes, preferably greater than about one hour, more preferably greaterthan about two hours, and even more preferably greater than about fourhours, eight hours, twelve hours or longer. Although a compound isstable using this criteria, physiological stability can also be measuredby observing the duration of biological effects on the patient. Theseeffects include amelioration or elimination of patient symptoms, anincrease in number or appearance of hemoglobin producing cells, or analteration, activation or suppression of specific gene expression, suchas, for example, the persistence of fetal globin chain expression inblood cells.

[0088] Symptoms may be clinically observed or biologically quantified.For example, observed symptoms are those which can be clinicallyperceived and include pathological alterations in cellular morphologysuch as red cell sickling, anemic crises, jaundice, splenomegaly,hepatomegaly, hemorrhaging, tissue damage due to hypoxia, organdysfunction, pain such as angina pectoris, fatigue including shortnessof breath, weakness and poor exercise ability, and pallor. Clinicalsymptoms which are important from the patient's perspective include areduced frequency or duration, or elimination of the need fortransfusions or chelation therapy. Quantifiable biological symptoms arethose which can be more accurately measured such as anemia, enzymeactivity, hematocrit and hemoglobin levels, decreased cell viability,ineffective erythropoiesis, abnormal reticulocyte count, abnormal ironloads, inadequate peripheral blood flow, anuria, dyspnea, hemolysis andspecific gene expression. Other quantifiable biological activitiesinclude, for example, the ability to recruit and stimulate theproliferation of hemoglobin expressing cells, the ability to increasehemoglobin expression, the ability to balance α-type and β-type globingene expression or the ability to increase expression of embryonic,fetal or at least partially functional pseudo-globin genes. Preferably,a stable compound of the invention has an in vivo half-life of greaterthan about 15 minutes, a serum half-life of greater than about 15minutes, or a biological effect which continues for greater than 15minutes after treatment has been terminated or the serum level of thecompound has decreased by more than half.

[0089] Compositions are not significantly biotransformed, degraded orexcreted by catabolic processes associated with metabolism. Althoughthere may be some biotransformation, degradation or excretion, thesefunction are not significant if the composition is able to exert itsdesired effect. Catabolic processes include deamination of aminases,hydrolysis of esters and amides, conjugation reactions with, forexample, glycine or sulfate, oxidation by the cytochrome p450oxidation/reduction enzyme system and degradation in the fatty acidpathway. Hydrolysis reactions occur mainly in the liver and plasma by avariety of non-specific hydrolases and esterases. Both deaminases andamidases, also localized in the liver and serum, carry out a large partof the catabolic process. Reduction reactions occur mainlyintracellularly in the endoplasmic reticulum and transferases performconjugation reactions mainly in the kidneys and liver.

[0090] Compositions are also preferably safe at effective dosages. Safecompositions are compositions that are not substantially toxic (e.g.cytotoxic or myelotoxic), or mutagenic at required dosages, do not causeadverse reactions or side effects, and are well tolerated. Although sideeffects may occur, compositions are substantially safe if the benefitsachieved from their use outweigh disadvantages that may be attributableto side effects. Unwanted side effects include nausea, vomiting, hepaticor renal damage or failure, hypersensitivity, allergic reactions,cardiovascular problems, gastrointestinal disturbances, seizures andother central nervous system difficulties, fever, bleeding orhemorrhaging, serum abnormalities and respiratory difficulties.

[0091] Compositions useful for treating blood disorders preferably donot substantially affect the viability of a cell such as a normalmammalian cell, the cell being treated or effected by the chemicalcompound. Normal cell viability, the viability of an untransformed oruninfected cell, can be determined from analyzing i Ad the effects ofthe composition on one or more biological processes of the cell.Detrimental interference with one or more of these cellular processesbecomes significant when the process becomes abnormal. Examples ofquantitatable and qualifiable biological processes include the processesof cell division, protein synthesis, nucleic acid (DNA or RNA)synthesis, nucleic acid (principally DNA) fragmentation and apoptosis.Others processes include specific enzyme activities, the activities ofthe cellular transportation systems such as the transportation of aminoacids by system A (neutral), system B (acidic) or system C (basic), andthe expression of a cell surface protein. Each of these parameters iseasily determined as significantly detrimental, for example, in tissueculture experiments, in animal experiments or in clinical studies usingtechniques known to those of ordinary skill in the art. Abnormal celldivision, for example, can be mitosis which occurs too rapidly, as in amalignancy, or unstably, resulting in programmed cell death orapoptosis, detected by increased DNA degradation. The determination ofabnormal cell viability can be made on comparison with untreated controlcells. Compositions preferably increase normal cell viability. Increasedcell viability can be determined by those of ordinary skill in the artusing, for example, DNA fragmentation analysis. A decreased amount offragmentation indicates that cellular viability is boosted.Determinations of increased or decreased viability can also be concludedfrom an analysis of the results of multiple different assays. Wheremultiple tests provide conflicting results, accurate conclusions canstill be drawn by those of ordinary skill based upon the cell type, thecorrectness or correlation of the tests with actual conditions and thetype of composition.

[0092] Compositions can be prepared in solution as a dispersion,mixture, liquid, spray, capsule or as a dry solid such as a powder orpill, as appropriate or desired. Solid forms may be processed intotablets or capsules or mixed or dissolved with a liquid such as water,alcohol, saline or other salt solutions, glycerol, saccharides orpolysaccharide, oil or a relatively inert solid or liquid. Liquidsadministered orally may include flavoring agents such as mint, cherry,guava, citrus, cinnamon, orange, mango, or mixed fruit flavors toincrease palatability. Pills, capsules or tablets administered orallymay also include flavoring agents. Additionally, all compositions mayfurther comprise agents to increase shelf-life, such as preservatives,anti-oxidants and other components necessary and suitable formanufacture and distribution of the composition. Compositions furthercomprise a pharmaceutically acceptable carrier. Carriers are chemical ormulti-chemical compounds that do not significantly alter or effect theactive ingredients of the compositions. Examples include water, alcoholssuch as glycerol and polyethylene glycol, glycerin, oils, salts such assodium, potassium, magnesium and ammonium, fatty acids, saccharides orpolysaccharides. Carriers may be single substances or chemical orphysical combinations of these substances.

[0093] Another embodiment of the invention is directed to combinationsof compositions comprising a chemical compound in combination with anagent know to positively affect hemoglobin expression or hemoglobinexpressing cells. The agent may be a chemical compound such as aceticacid, butric acid, D- or L-amino-n-butyric acid, α- or β-amino-n-butricacid, arginine butyrate or isobutyramide, all disclosed in U.S. PatentNos. 4,822,821 and 5,025,029. Others include butyrin, 4-phenyl butyrate(C₆H₅CH₂CH₂CH₂COOH), phenylacetate (C₆H₅CH₂COOH), phenoxy acetic acid,all of which and more are disclosed in U.S. Pat. No. 4,704,402, and U.S.patent application Ser. No. 08/398,588 (entitled “Compositions for theTreatment of Blood Disorders” filed Mar. 3, 1995), and derivatives,salts and combination of these agents. Alternatively, the agent may be ahematopoietic protein such as erythropoietin, steel factor, insulin, aninterleukin, a growth factor, hormones such as activin or inhibin,disclosed in U.S. Pat. Nos. 5,032,507 and 4,997,815, and activefragments and combinations of these proteins either with each other orwith other chemical compounds. Such composition may have additive orsynergistic effects.

[0094] Another embodiment of the invention is directed to methods forthe treatment of patients with blood disorder comprising the pulsedadministration of one or more compositions. Compositions to beadministered contain a therapeutically effective pulsed amount of achemical compound or proteinaceous agent. A therapeutical effectivepulsed amount is that amount which has a beneficial effect to thepatient by alleviating one or more symptoms of the disorder or simplyreduce premature mortality. For example, a beneficial effect may be adecrease in pain, a decrease in duration, frequency or intensity ofcrises, an increased hematocrit, an improved erythropoiesis, a reducedor eliminated necessity for chelation therapy, an increased reticulocytecount, an increased peripheral blood flow, a decreased hemolysis,decreased fatigue or an increased strength. Preferably, a therapeuticamount is that amount of chemical compound or agent that stimulates orenhances the expression of non-adult globin such as embryonic or fetalglobin, or the proliferation of embryonic, fetal or adult globinexpressing cells. A therapeutically effective amount for continuoustherapy is typically greater than a therapeutically amount that iseffective in pulsed therapy. Consequently, pulsed therapy exposes thepatient to lower levels of the composition and/or the active ingredientthan would be needed with non-pulse therapy.

[0095] Compositions provided to the patient may include any combinationof the proteins or chemical compounds described herein or known to thoseof ordinary skill in the art. The patient may be a domesticated animalsuch as a dog, cat, horse, cow, steer, pig, sheep, goat or chicken, or awild animal, but is preferably a human or another primate.Administration may be to an adult, an adolescent, a child, a toddler, aneonate or an infant, or administered in utero. Administration of thecomposition may be short term, continuous or sporadic as necessary.Patients with a suspected or diagnosed with a blood disorder may onlyrequire composition treatment for short periods of time or untilsymptoms have abated or have been effectively eliminated.

[0096] Compositions can be directly or indirectly administered to thepatient. Indirect administration is performed, for example, byadministering the composition to cells ex vivo and subsequentlyintroducing the treated cells to the patient. The cells may be obtainedfrom the patient to be treated or from a genetically related orunrelated patient. Related patients offer some advantage by lowering theimmunogenic response to the cells to be introduced. For example, usingtechniques of antigen matching, immunologically compatible donors can beidentified and utilized.

[0097] Direct administration of a composition may be by oral,parenteral, sublingual, rectal such as suppository or enteraladministration, or by pulmonary absorption or topical application.Parenteral administration may be by intravenous injection, subcutaneousinjection, intramuscular injection, intra-arterial injection,intrathecal injection, intra peritoneal injection or direct injection orother administration to one or more specific sites. Injectable forms ofadministration are sometimes preferred for maximal effect in, forexample, bone marrow. When long term administration by injection isnecessary, venous access devices such as medi-ports, in-dwellingcatheters, or automatic pumping mechanisms are also preferred whereindirect and immediate access is provided to the arteries in and aroundthe heart and other major organs and organ systems.

[0098] Another effective method of administering the composition is bytransdermal transfusion such as with a dermal or cutaneous patch, bydirect contact with, for example, bone marrow through an incision orsome other artificial opening into the body. Compositions may also beadministered to the nasal passages as a spray. Arteries of the nasalarea provide a rapid and efficient access to the bloodstream andimmediate access to the pulmonary system. Access to the gastrointestinaltract, which can also rapidly introduce substances to the blood stream,can be gained using oral, enema, suppository, or injectable forms ofadministration. Compositions may be administered as a bolus injection orspray. Compositions that may or may not be pulsed may be givensequentially over time (episodically) such as every two, four, six oreight hours, every day (QD) or every other day (QOD), or over longerperiods of time such as weeks to months. Compositions may also beadministered in a timed-release fashion such as by using slow-releaseresins and other timed or delayed release materials and devices.

[0099] Orally active compositions are more preferred as oraladministration is usually the safest, most convenient and economicalmode of drug delivery. Oral administration is usually disadvantageousbecause compositions are poorly absorbed through the gastrointestinallining. Compounds which are poorly absorbed tend to be highly polar.Consequently, compounds which are effective, as described herein, may bemade orally bioavailable by reducing or eliminating their polarity. Thiscan often be accomplished by formulating a composition with acomplimentary reagent which neutralizes its polarity, or by modifyingthe compound with a neutralizing chemical group. Oral bioavailability isalso a problem because drugs are exposed to the extremes of gastric pHand gastric enzymes. These problems can be overcome in a similar mannerby modifying the molecular structure to withstand very low pH conditionsand resist the enzymes of the gastric mucosa such as by neutraing anionic group, by covalently bonding an ionic interaction, or bystabilizing or removing a disulfide bond or other relatively labilebond.

[0100] Treatments to the patient may be therapeutic or prophylactic.Therapeutic treatment involves administration of one or morecompositions of the invention to a patient suffering from one or moresymptoms of the disorder. Symptoms typically associated with blooddisorders include, for example, anemia, tissue hypoxia, organdysfunction, abnormal hematocrit values, ineffective erythropoiesis,abnormal reticulocyte count, abnormal iron load, splenomegaly,hepatomegaly, impaired peripheral blood flow, dyspnea, increasedhemolysis, jaundice, anemic crises and pain such as angina pectoris.Relief and even partial relief from one or more of these symptomscorresponds to an increased life span or simply an increased quality oflife. Further, treatments that alleviate a pathological symptom canallow for other treatments to be administered.

[0101] Prophylactic treatments involve pulsed administration of acomposition to a patient having a confirmed or suspected blood disorderwithout having any overt symptoms. For example, otherwise healthypatients who have been genetically screened and determined to be at highrisk for the future development of a blood disorder may be administeredcompositions of the invention prophylactically. Administration can beginat birth and continue, if necessary, for life. Both prophylactic andtherapeutic uses are readily acceptable because these compounds aregenerally safe and non-toxic.

[0102] Another embodiment of the invention is directed to a method forregulating the expression of α globin gene in a mammalian cell. Briefly,the cell is exposed to an effective amount of a composition. A poorlyexpressed or quiescent globin gene of the cell is stimulated to increasethe expression of its protein product. An effective amount of thecomposition is that amount which increases the extent or magnitude ofhematopoiesis, increases the proliferation of hemoglobin expressingcells, increases, decreases or balances expression from one or moreglobin genes, or increases or stimulates the specific expression of oneor more globin genes such as an alpha (α) globin gene, a zeta (ζ) globingene, an epsilon (ε) globin gene, a beta (β) globin gene, a delta (δ)globin gene, α gamma (G-γ or A-γ) globin gene, or an, at least, partlyfunctional pseudo-globin gene. Cells can be treated in culture or invivo. Cultures of treated cells will produce increased amounts ofhemoglobin and preferably embryonic or fetal globin. This hemoglobin canbe harvested for introduction to a patient or the stimulated cellsthemselves can be administered to the patient. Alternatively,recombinant cells containing α globin gene which can be stimulated bycompositions of the invention can be utilized. These recombinant cellsmay be heterologous or homologous natural cells, or syntheticallycreated cells such as a lipid vesicles.

[0103] Another embodiment of the invention is directed to a method forregulating the proliferation of red blood cells and, preferably,specifically regulating the expression of fetal hemoglobin. As above, aneffective amount of a composition is administered in pulses to, forexample, a cell population obtained from stem cells, bone marrow, cordblood, yolk sac cells, or fetal cells such as fetal liver cells, orcombinations thereof, ex vivo. The pulse-treated cells, or purifiedproducts harvested from these cells, are then administered to a patientin vivo. This method can be utilized to treat blood disorders inpatients by increasing the amount of one or more different types ofglobin or hemoglobin expressing cells can alleviate symptoms associatedwith a blood disorder. Cells can be obtained from volunteers or thepatients to be treated. Alternatively, treated cells or products derivedfrom treated cells can be harvested, purified by, for example, columnchromatography, and utilized for other medical applications such asdiagnostic or other treatment monitoring screening kits.

[0104] Another embodiment of the invention is directed to a method forameliorating a blood disorder by administering a therapeuticallyeffective amount of a pharmaceutical composition containing an agentthat stimulates the expression of α globin gene or stimulates theproliferation of hemoglobin expressing cells wherein the compositiondoes not significantly decrease viability of the cell being treated or anormal cell. The therapeutically effective amount is that amount whichameliorates one or more symptoms of the blood disorder or reducespremature mortality. A normal cell is a relatively healthy mammaliancell that is not otherwise infected or transformed. Viability can beassayed by determining the effect of the composition on cell division,protein or nucleic acid synthesis, biochemical salvage pathways, aminoacid or nucleotide transport processes, nucleic acid fragmentation orapoptosis and comparing the effects observed to control cells. Pulsing,according to the described treatment regimens, can also be used toadminister these and other compositions of the invention and theireffects tested in tissue culture, in vivo or by cell counting.

[0105] Patients with blood disorders are typically quite infirm with,for example, iron damaged organs and systems. Most treatments furthertax the patient's already frail health in an effort to combat thedisorder. This is true for both arginine butyrate and isobutyramidewhich decrease cell viability as determined in DNA fragmentation assays.To decrease cell viability is not desired for the treatment of blooddisorders and may even be harmful. Surprisingly, many of the pulsedcompositions maintain or, preferably, increase cell viability. This is agreat benefit in the treatment of blood disorders and can significantlyincrease thee chances for a successful outcome for the patient. Forexample, the pulsed administration of phenoxyacetic acid or butyric acidethyl ester both reduce DNA fragmentation in fragmentation assays, andphenoxyacetic acid and α-methyl hydrocinnamic acid do not significantlyalter system A transport of amino acids.

[0106] As such, pulsed composition can be used to treat or prevent ironoverloaded or iron deficient systems such as occurs in transfusedpatients and anemic patients with thalassemia or sickle cell anemia. Aschemicals of the compositions of the invention regulate systems thatexploit iron, the amount of free and the amount of available iron in apatient's system can be regulated and carefully controlled. Chelationtherapy, often the only conventional treatment available for ironover-loaded transfusion patients, may be lessened or avoided entirely.As chelation therapy is often uncertain and with some risk of its own,the long-term prognosis for these patients is greatly improved.

[0107] Another embodiment of the invention is directed to a method forincreasing fetal hemoglobin comprising the pulsed administration of acomposition to a patient. For example, hemoglobin F content of blood sotreated is increased greater than about 2%, preferably greater thanabout 5% and more preferably greater than about 10%. Patients which canbe treated include any mammal such as a human. Chemical compounds whichcould be utilized include C₁-C₄ substituted and phenyl substitutedphenoxy acetic acid, C₁-C₄ substituted and phenyl substituted cinnamicacid, C₁-C₄ substituted and/or phenyl substituted hydrocinnamic acid,α-methyl hydrocinnamic acid, C₁-C₄ substituted and phenyl substitutedacetic acid, C₁-C₄ substituted and phenyl substituted propionic acid,and C₁-C₄ substituted and/or phenyl substituted butyric acid, or aderivative or modification thereof. Such methods are useful to treat orprevent blood disorders in the same or a different patient. For example,to treat the same patient, the compound can be pulse administered for atherapeutically effective period of time to allow the hemoglobin contentof just the globin protein content to rise. Alternatively, the patientcan be treated and the patient's blood collected at peak times ofhemoglobin or globin production, collected and stored, and administeredto another patient or re-administered to the same patient. Suchtreatments would be useful therapies for those being treated withradiator therapy, chemotherapy, bone marrow transplants, blood diseases,such as sickle cell disease and thalassemia, and other disorders whichwould be alleviated with an increased blood hemoglobin content.

[0108] Another embodiment of the invention is directed to methods forthe treatment of a patient with an infection or a neoplastic disordercomprising the pulsed administration of a therapeutically effectivecomposition. Treatable infectious diseases include bacterial infectionssuch as sepsis and pneumonia, infections caused by bacterial pathogenssuch as, for example, Pneumococci, Streptococci, Staphylococci,Neisseria, Chlamydia, Mycobacteria, Actinomycetes and the entericmicroorganisms such as enteric Bacilli; viral infections caused by, forexample, a hepatitis virus, a retrovirus such as HIV, an influenzavirus, a papilloma virus, a herpes virus (HSV I, HSV II, EBV), a polyomavirus, a slow virus, paramyxovirus and corona virus; parasitic diseasessuch as, for example, malaria, trypanosomiasis, leishmania, amebiasis,toxoplasmosis, sarcocystis, pneumocystis, schistosomiasis andelephantitis; and fungal infections such as candidiasis,phaeohyphomycosis, aspergillosis, mucormycosis, cryptococcosis,blastomycosis, paracoccidiodomycosis, coccidioidomycosis, histomycosis,actinomycosis, nocardiosis and the Dematiaceous fungal infections.

[0109] Anti-neoplastic activity includes, for example, the ability toinduce the differentiation of transformed cells including cells whichcomprise leukemias, lymphomas, sarcomas, neural cell tumors, carcinomasincluding the squamous cell carcinomas, seminomas, melanomas,neuroblastomas, mixed cell tumors, germ cell tumors, undifferentiatedtumors, neoplasm due to infection (e.g. viral infections such as a humanpapilloma virus, herpes viruses including Herpes Simplex virus type I orII or Epstein-Barr virus, a hepatitis virus, a human T cell leukemiavirus (HTLV) or another retrovirus) and other malignancies. Upondifferentiation, these cells lose their aggressive nature, no longermetastasize, are no longer proliferating and eventually die and/or areremoved by the T cells, natural killer cells and macrophages of thepatient's immune system. The process of cellular differentiation isstimulated or turned on by, for example, the stimulation and/orinhibition of gene specific transcription. Certain gene products aredirectly involved in cellular differentiation and can transform anactively dividing cell into a cell which has lost or has a decreasedability to proliferate. An associated change of the pattern of cellulargene expression can be observed. To control this process includes theability to reverse a malignancy. Genes whose transcriptional regulationare altered in the presence of compositions of the invention include theoncogenes myc, ras, myb, jun, fos, abi and srcr The activities of thesegene products as well as the activities of other oncogenes are describedin J. D. Slamon et al. (Science 224:256-62, 1984).

[0110] Another example of anti-neoplastic activity includes the abilityto regulate the life cycle of the cell, the ability to repressangiogenesis or tissue regeneration through the blockade or suppressionof factor activity, production or release, the ability to regulatetranscription or translation, or the ability to modulate transcriptionof genes under angiogenesis, growth factor or hormonal control. Theseactivities are an effective therapy particularly againstprostatic-neoplasia and breast carcinomas. Additional anti-neoplasticactivities include the ability to regulate the cell cycle for example byeffecting time in and passage through S phase, M phase, G₁ phase or G₀phase, the ability to increase intracellular cAMP levels, the ability toinhibit or stimulate histone acetylation, the ability to methylatenucleic acids and the ability to maintain or increase intracellularconcentrations of anti-neoplastic agents.

[0111] The neoplastic disorder may be any disease or malady which couldbe characterized as a neoplasm, a tumor, a malignancy, a cancer or adisease which results in a relatively autonomous growth of cells.Neoplastic disorders prophylactically or therapeutically treatable withcompositions of the invention include small cell lung cancers and otherlung cancers, rhabdomyosarcomas, chorio carcinomas, glioblastomamultiformas (brain tumors), bowel and gastric carcinomas, leukemias,ovarian cancers, prostate cancers, osteosarcomas or cancers which havemetastasized. Diseases of the immune system which are treatable by thesecompositions include the non-Hodgkin's lymphomas including thefollicular lymphomas, Burlitt's lymphoma, adult T-cell leukemias andlymphomas, hairy-cell leukemia, acute myelogenous, lymphoblastic orother leukemias, chronic myelogenous leukemia, and myelodysplasticsyndromes. Additional diseases treatable by the compositions includevirally -induced cancers wherein the viral agent is EBV, HPV, HIV, CMV,HTLV-1 or HBV, breast cell carcinomas, melanomas and hematologicmelanomas, ovarian cancers, pancreatic cancers, liver cancers, stomachcancers, colon cancers, bone cancers, squamous cell carcinomas,neurofibromas, testicular cell carcinomas and adenocarcinomas.

[0112] In another embodiment of the invention, compositions may be pulseadministered in combination with other anti-neoplastic agents ortherapies to maximize the effect of the compositions in an additive orsynergistic manner. Cytokines which may be effective in combination withthe compositions include growth factors such as B cell growth factor(BCGF), fibroblast-derived growth factor (FDGF), granulocyte/macrophagecolony stimulating factor (GM-CSF), granulocyte colony stimulatingfactor (G-CSF), macrophage colony stimulating factor (M-CSF), epidermalgrowth factor (EGF), platelet derived growth factor (PDGF) nerve growthfactor (NGF), stem cell factor (SCF), and transforming growth factor(TGF). These growth factors plus a composition may further stimulatecellular differentiation and/or the expression of certain MHC antigensor tumor specific antigens. For example, BCGF plus a composition may beeffective in treating certain B cell leukemias. NGF plus a compositionmay be useful in treating certain neuroblastomas and/or nerve celltumors. In a similar fashion, other agents such as differentiatingagents may be useful in combination with a composition to prevent ortreat a neoplastic disorder. Other differentiating agents include B celldifferentiating factor (BCDF), erythropoietin (EPO), steel factor,activin, inhibin, the bone morphogenic proteins (BMPs), retinoic acid orretinoic acid derivatives such as retinol, the prostaglandins, and TPA.

[0113] Alternatively, other cytokines and related antigens incombination with a composition may also be useful to treat or preventneoplasia. Potentially useful cytokines include tumor necrosis factor(TNF), the interleukins (IL-1, IL-2, IL-3, etc.), the interferonproteins (IFN) IFN-α, IFN-γ, and IFN-γ, cyclic AMP including dibutyrylcyclic AMP, hemin, hydroxyurea, hypoxanthine, glucocorticoid hormones,dimethyl sulfoxide (DMSO), and cytosine arabinoside, and anti-viralssuch as acyclovir and gemciclovirs. Therapies using combinations ofthese agents would be safe and effective against malignancies and otherforms of cancer. Combinations of therapies may also be effective ininducing regression or elimination of a tumor or some other form ofcancer such as pulsed compositions plus radiation therapy, toxin or drugconjugated antibody therapy using monoclonal or polyclonal antibodiesdirected against the transformed cells, gene therapy or specificanti-sense therapy. Effects may be additive, logarithmic, orsynergistic, and methods involving combinations of therapies may besimultaneous protocols, intermittent protocols or protocols which areempirically determined.

[0114] Another embodiment of the invention comprises methods for thepulse administration of compositions for the treatment of neoplasticdisorders by augmenting conventional chemotherapy, radiation therapy,antibody therapy, and other forms of therapy. Compositions containingchemical compounds in combination with chemotherapeutic agents, enhancethe effect of the chemotherapeutic agent alone. Compositions decreasethe expression or activity of proteins responsible for lowering theintra -cellular concentration of chemotherapeutic agents. Proteinsresponsible for resistance to drugs and other agents, the multi-drugresistance (MDR) proteins, include the β-glycoprotein (Pgp) encoded bythe mdr-1 gene. Consequently, conventional drugs for the treatment ofneoplastic disorders accumulate at higher concentrations for longerperiods of time and are more effective when used in combination with thecompositions herein. Some conventional chemotherapeutic agents whichwould be useful in combination therapy with compositions of theinvention include the cyclophosphamide such as alkylating agents, thepurine and pyrimidine analogs such as mercapto-purine, the vinca andvinca -like alkaloids, the etoposides or etoposide like drugs, theantibiotics such as deoxyrubocin and bleomycin, the corticosteroids, themutagens such as the nitrosoureas, antimetabolites includingmethotrexate, the platinum based cytotoxic drugs, the hormonalantagonists such as antiinsulin and antiandrogen, the antiestrogens suchas tamoxifen an other agents such as doxorubicin, L-asparaginase,dacarbazine (DTIC), amsacrine (mAMSA), procarbazine, hexamethylmelamine,and mitoxantrone. The chemotherapeutic agent could be givensimultaneously with the compounds of the invention or alternately asdefined by a protocol designed to maximize drug effectiveness, butminimize toxicity to the patient's body.

[0115] Another embodiment of the invention is directed to aids for thetreatment of human disorders such as infections, neoplastic disordersand blood disorders. Aids contain compositions of the invention inpredetermined amounts which can be individualized in concentration ordose for a particular patient. Compositions, which may be liquids orsolids, are placed into reservoirs or temporary storage areas within theaid. At predetermined intervals, a set amount of one or morecompositions are administered to the patient. Compositions to beinjected may be administered through, for example, mediports orin-dwelling catheters. Aids may further comprise mechanical controls orelectrical controls devices, such as a programmable computer or computerchip, to regulate the quantity or frequency of administration topatients. Examples include both single and dual rate infusers andprogrammable rinsers. Delivery of the composition may also be continuousfor a set period of time. Aids may be fixed or portable, allowing thepatient as much freedom as possible.

[0116] The following examples are offered to illustrate embodiments ofthe present invention, but should not be viewed as limiting the scope ofthe invention.

EXAMPLES

[0117] Treatment of K562 Cells and Analysis of Globin mRNA

[0118] K562 cells kindly provided by Dr. George Atweh were cultured with10% fetal bovine serum (Sigma, St. Louis, Mo.) and RPMI media (GrandIsland Biological Company, Grand Island, New York) in a humidifiedatmosphere with 5% CO2/95% air. Compounds were tested at a finalconcentration of 1 mM at neutral pH and included butyric acid,phenoxyacetic acid, dimethylbutric acid, alpha methylhydrocionamic acid,2,3, and 4-methoxyhydrocinnamic acid, dihydrocinnamic acid,methoxycinnamic acid, methoxyacetic acid, phenylpropionic acid, aminohydrocinnamic acid, DL β- and DL-βamino-n-butyric acid, cinnamic acid,and 2 methylhydrocinnamic acid (Aldrich Chemical Company, St. Louis,Mo.). Additional compounds studied included dimethylhydroxy acetic acid,dimethylpropionic acid, dimethylphenoxyacetic acid, anddimethylmethoxyacetic acid. After three days of culture with theseagents, mRNA was purified and α, β, and γ globin mRNA was analyzed byprimer extension using oligonucleotide primers and quantitation on aPhosphoImager as previously described. A representative autoradiogramand a summary of the globin expression induced by the effectivecompounds is shown in FIG. I and Table I.

[0119] Proliferation Studies Using 32D Cells

[0120] 32D cells were cultured in RPMI media with 10% fetal bovine serum(Sigma, St. Louis, Mo.), 100 mM glutamine (GIBCO), and murine IL3 (20U/ml) (Biosource International). Growth factor controls used includedthe standard concentration of IL-3 required for proliferation of thesecells (25 U/ml) and a 50-fold lower concentration (0.5 U/ml), anderythropoietin (3 U/ml) or G-CSF ( U/ml), (Amgen, Thousand Oaks,Calif.). The test compounds were added at final concentrations of 1 mM.As a cell density of 2.5−10×10⁵ is necessary for growth of this cellline, this density was maintained by passing the cells at three dayintervals or by concentrating the cells when apoptosis occurred.Proportions of cells which were viable or apoptotic, and the fraction ofcells in each part of the cell cycle was assessed by incubating thecells with Trypan blue and enumeration, and with propidium iodideincubation and FACScan analysis as previously described.

[0121] In Vivo Administration in Mice

[0122] To determine if a prototype test compound has in vivo activity instimulating erythropoeisis, methylhydrocinnamic acid was administered toC₅₇ black mice. Mice were cared for and experiments were performedaccording to regulations of the Committee on Animal Research at theUniversity of Southern Alabama. The test compound was administered byintraperitoneal injection three times per date for seven days at a totaldaily dose of 300 mg/kg. Blood (50 μl) was sampled from theretro-orbital space and reticulocytes were quantitated by staining with1% brilliant cresyl blue and counting the percentage of reticulumpositive cells in 1000 cells. Reticulocytes were computed to controlmice which were injected with the same volume of normal saline and whichreceived a 50 μl daily phlebotomy for twenty-one days without asignificant change in hematocrit or a significant increase inreticulocyte counts (B. Pace, unpublished observations).

[0123] Phamrucokinetic Studies

[0124] Baboons were cared for according to regulations of the Committeeon Animal Care at the University of Oklahoma Health Sciences Center.Chronic indwelling venous and arterial catheters which were maintainedusing sterile technique for blood sampling. Compounds were administeredby nasogastric tube and blood was collected to determine drug plasmalevels at regular intervals following single oral doses. Three doses ofone compound were also studied in two human volunteers. The testcompounds were analyzed after ether extraction of the plasma, separationby HPLC, and quantitated by comparison to a spilled internal standard ofheptanoic acid according to previously described methods.

[0125] The effects of the representative compounds which have beensynthesized or selected for resistance to beta oxidative metabolism andglucuronidation in stimulating γ globin gene expression in a humanerythroid-like cell line and for their effects on cell growth utilizinga multi-lineage murine hematopoietic cell line, 32D. This cell line isdependent on high concentrations of IL-3 for growth. 32D cells undergoapoptotic cell death if IL-3 is completely withdrawn and do notproliferate when IL-3 concentrations are reduced by 50-fold over thelevels required for proliferation. No condition or growth factor hasbeen found to abrogate the IL-3 dependency of this cell line for cellproliferation (Patel, Oncogene 13:1197 (1996)). In the presence of IL3depletion, these cells also terminally differentiate along the erythroidlineage in the presence of erythropoietin or terminally differentiateinto mature granulocytes in the presence of G-CSF. Some test compoundswhich stimulated γ globin expression also supported proliferation ofthis multi-lineage cell line and prevented apoptotic cell death whenIL-3 was withdrawn. In vivo activity was also found with a prototypetest compound administered mice. Finally. half-lives for three prototypecompounds were found to be several hours following oral administrationto baboons, demonstrating potential therapeutic utility.

RESULTS

[0126] Effects of the test compounds on globin gene expression wereassessed by comparing the ratios of I globin:a globin mRNA and the ratioof γ globin mRNA in treated cells were compared to γ globin MRNA incontrol cells, adjusted for an internal control. γ globin mRNA increasedby 2.4 to 26-fold over untreated (control) K562 cells in the presence ofseveral of the test compounds, as shown in Table I. The most activecompounds in stimulating γ globin compared to control cells werephenoxyacetic acid, 2-methylhydrocinnamic acid and α-methylhydrocinnamicacid, 2-methoxycinnamic acid, dimethoxyphenyl acetic acid, butyrate, and2,2-dimethylbutyrate. These results are consistent with previous findingthat these and similar compounds stimulate γ globin expression inerythroid progenitors cultured from human subjects and from CD34+ cellsisolated from fetal liver.

[0127] Under culture conditions containing recombinant murine IL-3 at 50U/ml, the optimal concentrations for cell proliferation, apoptosis wasdetected in less than 10% of the cell population and 32D cells doubledafter 3 days. Apoptosis in 32D cells increased to 80% when IL-3 levelswere decreased by 50-fold, from 25 U/ml to 0.5 U/ml. The cells underwent100% apoptosis in the complete absence of IL-3 (FIG. 2). In contrast,when IL-3 was decreased to 0.5 U/ml, the minimum required to preventapoptosis, cell numbers did not significantly, increase and plateauedafter 2 days. In the presence of 0.5 U/ml IL-3 and addition oferythropoietin or G-CSF, cell proliferation occurred along the erythroidand myeloid pathways respectively as has been previously reported, andcell numbers increased by 2-3 fold over 5 days, shown in FIG. 2. In thepresence of phenoxyacetic acid, alpha methylhydrocinnamic acid,dimethylbutyric acid, DL-βamino-n-butyric acid and dimethylhydroxyaceticacid, however, cell proliferation increased 2 to 3-fold despite the lowconcentration of IL-3 (FIG. 2). In contrast, addition of 1 mM butyratewith the low concentration of IL-3 resulted in cell death. Addition of 1mM test compounds with the same low concentration of IL-3 resulted in a2.5-3-fold increase in cell proliferation with several compounds abovethat observed with the marginal IL-3 concentration alone and resulted ina degree of proliferation similar to that induced by erythropoietin andG-CSF.

[0128] Bioavailability and Pharmacokinetic studies of certain testcompounds were performed in juvenile baboons using oral delivery of thetest compounds via gavage. Millimolar plasma levels were detectedfollowing single oral doses of phenoxyacetic acid, dimethylbutyrc acid,and methylhydrocinnamic acid and these levels persisted for 6 hours orlonger. Calculated half-lives were 6.5, 6.8, and 7.6 hours respectively,following doses of 100-500 mg/kg. These peak plasma levels are higherthan the concentration of compound which was required for γ globinstimulation in primary hematopoietic cells in vitro.

[0129] To determine how general the effects of these compounds may be,one lead compound, alpha methylhydrocinnamic acid was also administeredto mice. Administration of the compound resulted in a 200-600% (2-6fold) increase in reticulocytes over baseline. Reticulocytosis wasobserved in a step-wise manner and in a time-frame consistent with thetime required for development and maturation of late and early murineerythroid progenitors (3 and 6 days, respectively). Reticulocytesincreased by only 6-8% after 21 days of saline-injections in controlmice phlebotomized to the same (50 μl/day) degree. Hematocrits did notchange in controls over this time (B. Pace, unpublished observations).

[0130] Cell proliferation stimulation is transgenic mice, baboons, humancell culture, and a murine multi-lineage cell line by the activecompounds, genes whose expression is increased early in cellproliferation induced by hematopoietic growth factors such as IL-3 anderythropoietin were examined. RNA was extracted from 32D cells treatedwith the compounds for one day and for 11 days. Northern blots wereprepared with probes for the early growth related genes c-myb and c-mycand beta actin and histone H₃ were used as controls. Increasedexpression of c-myb occurs transiently, and early, when growth isinduced by erythropoietin and IL-3. See FIG. 5.

[0131] Of multiple compounds tested, c-myb was induced by 3-4 fold bythe compounds methylhydrocinnamic acid, dimethylbutyric acid,phenoxyacetic acid, DL-beta and D-alpha-amino butyric acid, 2,2-dimethylmethoxyacetic acid, and dimethyl propionic acid (alpha dimethylhydrocinnamic acid). C-myb was induced 2-fold with beta aminohydrocinnamic acid. The growth-related gene c-myc was induced 2-fold bythe same active compounds. Actin and histone H₃ mRNAs were not affectedby the compounds. See FIGS. 5 and 6.

[0132]FIG. 11 shows the relative steady-state accumulation of c-myb,c-myc, histone-3, and beta-actin mRNA in IL3-dependent 32D cells atdifferent time points after exposure of cells to different testcompounds. The first lane is from cells cultured in no IL-3, lane 2 in25 U/ml murine IL3 and lanes 3-18 have low IL-3 concentration (0.5 U/ml)plus test compounds. In addition, cells in lane 4 were treated with 100U/ml G-CSF, lane 5 2,2-dimethyl-methoxy acetic acid, lane 6 alphamethylhydrocinnamic acid, lane 7 phenoacetic acid, lane 8 argininebutyrate day 1 and 5, lane 9 α-dimethyl hydroacetic acid, lane 102,2-dimethylbutyric acid, lane 11 beta aminohydrocinnamic acid, lane 122-2-dimethylpropionic acid, lane 13 dimethylhydroxy acetic acid/a-methyllactic acid, lane 14 2-2-dimethylphenoxy acetic acid, lane 15 2,2dimethyl-l-phenoxyacetic acid, lane 16 cis-2 methoxy cinnamic acid, lane17 thioctic acid days 1 and 5, and lane 184-chlorophenoxy-2-propionic-acid days 1 and 5. All compounds were testedhere at 1 mM. Each set of treated cells is denoted by one numbered andone unnumbered lane consisting of mRNA from the same cells treated fordays 1 and 11 respectively, except where cells did not survive to day 11and only day 1 of treatment is shown. 20 ug of total RNA from eachsample were subjected to Northern blot analysis using specific probesfor c-myb, c-myc, actin, and histone H_(3.) One day and 11-day samplesfrom the same treated cells were quantitated by PhosphoImager.

[0133] In Vivo Experiments in Mice Transgenicfor the Human Beta GlobinGene Locus.

[0134] Three prototype compounds, methylhydrocinnamic acid (MHCA),phenoxyacetic acid (PAA), and dimethylbutyric acid PMB), wereadministered at doses from 100 to 250 mg/kg in two daily doses byintraperitoneal injection to mice transgenic for a human beta globinlocus YAC containing a silenced gamma globin gene. Reticulocytes, newlysynthesized red blood cells, were counted daily and non-alpha globin inMRNA was analyzed by Rnase protection. Only 50 microliters of blood wereremoved daily for testing. A 5 to 10-fold increase in reticulocytes anda 1.7-2.4 fold increase in gamma globin mRNA was observed within oneweek of therapy with the three protype compounds. In contrast, controlmice to which normal saline was administered, with the same degree ofphlebotomy for testing, had no significant changes in reticulocytes orglobin in mRNA.

[0135] Mice have more rapid metabolic rates than do larger animals, suchas humans and these compounds are still active in mice. Furthermore,gamma 9fetal) globin has not been readily inducible by compounds such asalpha amino-n-butyric acid in these same mice. Accordingly, the resultsare significant. See the following table: Reticulocytes γ/γ + β mRNA(fold (fold Animal Day 0 Peak increase) Day 0 Peak increase) DMB-1 2.417.7 (7.3) 0.20 0.36 (1.8) DMB-2 4.2 21.3 (5.1) 0.17 0.31 (1.7) MHCA-12.9 17.9 (5.6) 0.33 0.80 (2.4) MHCA-2 2.3 23.3 (10.1) 0.14 0.18 (1.5)

[0136] Control mice, to which normal saline was similarly administered,had no changes in reticulocytes or globin mRNA. [Mice have a highermetabolic rate than do larger animals, and γ globin has not always beeninducible by rapidly metabolized butyrates in these mice.]

[0137]FIG. 12 shows increase in young, newly proliferating red bloodcells after treatment with phenoxy acetic acid in four transgenic mice.Each curette represents one animal. Reticulocytes increased from 2.5 to7-fold with the highest increase resulting from the higher dose.

[0138] Hematopoietic stimulation in a baboon by the compounds AMHCA isshown in FIG. 13. An increase in multiple blood cell lineages resultedwhen a prototype hemoldne compound (a methylhydrocinnamic acid) wasadministered for five days to an anemic baboon, which was beingphlebotomized 5% of its blood volume daily. An increase in both whiteblood cells and total hemoglobin was observed.

[0139] Mononuclear cells from patients with sickle cell disease orthalassemia trait were isolated on Ficoll Hypague, washed, and culturedin methylcellulose media with optimal concentrations of hematopoieticgrowth factors IL-3, GM-CSF, Stem Cell Factor, IL6, 3 U/mlEiythropoietin, insulin, bovine serum albumin, and 0.2-0.5 mMconcentrations of test compounds of derivatives of cinnamic acid andhydrocinnamic acid. An increase in numbers of erythroid colonies overand was observed compared to control cultures containing optimalconcentrations of growth factors alone. The following illustrates somerepresentative cultures: TABLE 1 Mean BFU-E/culture 4 cultures averaged% Increase (per 0.2 million cells) over control Control 192 2methylbutyric acid 297 55% 3,5 dimethoxy4-hydroxycinnamic 215 11% acidControl 272 Transcinnamic acid 322 18% Control 176 Alphamethylhydrocinnamic acid 223 32.4%   2 Methylhydrocinnamic acid 21220.5%   4 Methoxycinnamic acid 191 8.5% 

[0140] TABLE 2 Effect of Compounds on Fetal and Alpha Globin mRNAs inK562 Cells Radioactivity α Fetal Globin Alpha Globin Compound b (γ) (α)γ/α Control  915479 118789 7.7 Arginine butyrate 2176523 296132 7.3Phenoxyacetic acid 2755891 507148 5.4 α-Methylhydrocinnamic acid 1648056 92979 17.7 2,2-Dimethylbutyric acid 1697936 178751 9.5trans-2-Methoxycinnamic acid  957146  36751 26.0 2-Methylhydrocinnamicacid 1388899  89473 15.5 cis-2-Methoxycinnamic acid 2255627 105452 21.4(3,4-Dimethoxyphenyl)acetic acid 1206529 106875 11.33-(3,4-Dimethoxyphenyl)propionic 1858358 191985 9.7 acid(2,5-Dimethoxyphenyl)acetic acid 1240100  85941 14.4

DISCUSSION

[0141] Suppression or inhibition of erythropoiesis and generalhematopoiesis in a dose-dependent fashion can be limitations of butratesand hydroxyurea, respectively, in the treatment of thep-hemoglobinopathies. Further disadvantages of the butyrates as optimaltherapeutics include their extremely rapid metabolism in uivo. Thecurrent studies arose from a search to identify novel orally-bioavailable compounds with long in vivo half-lives, which induce yglobin gene expression without simultaneously inducing cell growtharrest. Extensive investigation of agents which affect hematopoiesisduring the past decade has focused on multipotential hematopoieticgrowth factors which stimulate proliferation of multiple lineages suchas IL-3 and GM-CSF, the lineage-specific growth factors erythropoietinand G-CSF, the differentiating agents DMSO, butyric acid, retinoic acid,and HMBA and inhibitory factors, such as TGF-β and IFN-γ. Previouscomparison of the effects of butyric acid, which inhibits erythroidproliferation and α amino-n-butrric acid, which slightly stimulateserythroid progenitor growth, suggested that compounds with slightmodifications may also modulate erythroid cell growth. The findingsherein demonstrate that several classes of simple compounds, withspecific modifications in structure, stimulate the proliferation ofhematopoietic cells and can decrease the requirements for themultipotential growth factor IL-3. Abrogation of IL-3 requirements hasnot been previously found. As these compounds diffuse into cells freelywithout requiring receptors and diffuse into mitochondria, the compoundslikely exert their growth stimulating activities through metabolicpathways as well as through traditional signaling pathways, and throughtranscriptional regulation of growth-related genes.

[0142] The pattern of globin gene stimulation induces in K562 cells bysome of these compounds is complex, in that certain compounds (butrricand phenoxyacetic acid) stimulated expression of both α and γ globinMRNA. This may represent an effect of inducing differentiation of thesecells or of inducing expression of different globin genes. Othercompounds (cis 2-methoxyhydrocinnamic acid) curiously decreasedexpression of α globin, which accentuated the K562 α thalassemicphenotype. Such an effect would not be deleterious in humanβ-thalassemia, and would be expected to improve overall globin chainbalance. Phenoxyacetic acid, derivatives of hydro-cinnamic and cinnamicacid, and dimethylbutyric acid induced I globin MRNA and cellularproliferation. Such compounds particularly merit further investigationfor future consideration as therapeutics of the beta thalassemias, asthe accelerated erythroid apoptosis characteristic of these diseasesseverely limits the time-frame during which any Hemoglobin F stimulantcan act to improve globin chain balance before cell death occurs.

[0143] Several of the compounds studied here do not undergo rapidmetabolism in vivo, as do the simple fatty acids. The phenoxyacetic andphenylalkylacids and the dimethylated carboxylic acid derivatives wereselected for their structural resistance to usual routes of metabolismin vivo. A prototype of these compounds, a methylhydrocinnamic acid, didindeed have activity in mice, and three prototype compounds hadprolonged half-lives in the baboon. This result is significant becausemice have higher rates of metabolism than do humans and because similardoses of butyrate were previously not effective in mice transgenic forthe human γ globin gene without previous treatment with 5-azacytidine orwhen given at much higher doses. These and similar compoundsparticularly with modifications at the fourth position of a phenyl ringand the 2,2 dimethyl substituted carboxylic acids, appear attractive ashematopoietic stimulants for all lineages and as fetalhemoglobin-inducing agents.

I claim:
 1. A method for treating a human cell proliferative disorder bystimulating cell growth, comprising administering to a patient in need apharmaceutically effective amount of a composition containing aneffective amount of a C₁-C₄ substituted and/or phenyl substitutedcarboxylic acid and pharmaceutically acceptable salts thereof, and apharmaceutically acceptable carrier or diluent, wherein said C₁-C₄moiety and said phenyl moiety can be substituted or unsubstituted,wherein said substituents are selected from the group consisting ofhydroxy, halogens, phenyl, thiol, mercapto and methylthiol.
 2. Themethod of claim 1 , wherein said composition is a C₁-C₄ substitutedcarboxylic acid.
 3. The method of claim 1 , wherein said composition isa dimethyl-substituted carboxylic acid.
 4. The method of claim 1 whereinthe cytopenia is a red or white blood cell anemia, a leukopenia or athrombocytopenia.
 5. The method of claim 1 wherein the disorder is ahemoglobinopathy.
 6. A method of reducing the amount of a growthstimulating compound that must be administered to a patient having acell proliferative disorder comprising administering an effective amountof a composition containing a C₁-C₄ substituted and/or phenylsubstituted compound, wherein said compound is selected from the groupconsisting of cinnamic acid, acetic acid, butyric acid and propionicacid, or a pharmaceutically acceptable salt thereof, in apharmaceutically acceptable carrier or diluert, wherein said C₁-C₄moiety and said phenyl moiety can be substituted or unsubstituted, andsaid substituents are selected from the group consisting of hydroxy,halogens, phenyl, thiol, mercapto and methyl thiol.
 7. The method ofclaim 6 , wherein the composition is a dimethyl substituted compound. 8.The method of claim 7 , wherein the compound is selected from the groupconsisting of acetic acid, phenoxyacetic acid, methoxyacetic acid,cinnamic acid, hydrocinnamic acid, butyric acid, and propionic acid. 9.The method of claim 1 wherein the composition is administered bydelivery of a therapeutically effective pulsed dose of said compositionover a period of time and the therapeutically effective pulsed dosecomprises less of the composition than a therapeutic continuous doseadministered over said period of time.
 10. The method of claim 1 whereinthe composition is administered by injection, infusion, instillation oringestion.
 11. The method of claim 9 wherein said pulsed dose has aninterval between each pulse from about 3 to about 21 days.
 12. Themethod of claim 1 wherein treatment stimulates the number of circulatingplatelet cells or white blood cells as determined from peripheral bloodcell counts.